KR20240132482A - Oncolytic vaccinia virus and checkpoint inhibitor combination therapy - Google Patents

Abstract

(i) a pharmaceutical combination comprising a replicating oncolytic vaccinia virus and (ii) an immune checkpoint protein inhibitor, as well as kits comprising the pharmaceutical combination and methods for treating and/or preventing cancer are provided.

Description

{ONCOLYTIC VACCINIA VIRUS AND CHECKPOINT INHIBITOR COMBINATION THERAPY}

Cross-reference to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 62/488,623, filed April 21, 2017, and U.S. Provisional Patent Application No. 62/550,486, filed August 25, 2017, under 35 U.S.C. § 119(e), which are incorporated herein by reference in their entireties.

Technical field of the present invention

The present invention relates generally to virology and medicine. In certain aspects, the present invention relates to a therapeutic combination comprising a replicative oncolytic vaccinia virus and an immunomodulatory agent.

Normal tissue homeostasis is a highly regulated process of cell proliferation and cell death. An imbalance in either cell proliferation or cell death can lead to a cancerous state. For example, cervical, kidney, lung, pancreatic, colorectal, and brain cancers are just a few examples of the many cancers that can occur. In fact, the incidence of cancer is so high that more than 500,000 deaths are attributed to cancer each year in the United States alone.

Replication-selective oncolytic viruses have potential for cancer therapy. These viruses can induce tumor cell death through direct replication-dependent and/or viral gene expression-dependent antitumor effects. However, tumor-induced immunosuppression and early clearance of the virus result in a weak tumor-specific immune response, limiting the potential of these viruses as cancer therapeutics.

Similarly, immune checkpoint inhibitors have shown some promise in treating certain cancers, but only a limited percentage of patients achieve an objective clinical response. There remains a need for improved cancer therapies.

The present inventors have found that concurrent administration of an immune checkpoint inhibitor and intratumorally administered replicating oncolytic vaccinia virus results in a synergistic anti-tumor effect in clinically relevant cancer models, wherein the agents are administered simultaneously for a first administration and preferably for multiple sequential administrations. Accordingly, in some embodiments, the present application provides a combination therapy for use in the treatment and/or prevention of cancer and/or the establishment of metastases in a mammal, comprising concurrently administering to the mammal (i) a replicating oncolytic vaccinia virus and (ii) an immune checkpoint inhibitor, wherein the oncolytic vaccinia virus is administered intratumorally to the mammal. In certain aspects, concurrent administration of the pharmaceutical combination partners to the mammal provides enhanced and even synergistic anti-tumor immunity compared to either treatment alone.

In some embodiments, the replicating oncolytic vaccinia virus is administered intratumorally, intravenously, intraarterially, or intraperitoneally. In some embodiments, the replicating oncolytic vaccinia virus is administered intratumorally. In some embodiments, the replicating oncolytic vaccinia virus is administered in an amount effective to induce expression of an immune checkpoint protein in the tumor. In some embodiments, the tumor does not express the immune checkpoint protein or expresses the immune checkpoint protein at relatively low levels prior to administration of the replicating oncolytic vaccinia virus. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to an immune checkpoint protein, preferably a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof.

In some embodiments, the immune checkpoint inhibitor of the combination inhibits an immune checkpoint protein selected from the group consisting of cytotoxic T-lymphocyte antigen-4 (CTLA4 or CTLA-4), programmed cell death protein 1 (PD-1), B7-H3, B7-H4, T-cell membrane protein 3 (TEM3), galectin 9 (GAL9), lymphocyte activation gene 3 (LAG3), V-domain immunoglobulin (Ig)-containing inhibitor of T-cell activation (VISTA), killer-cell immunoglobulin-like receptor (KIR), B and T lymphocyte attenuator kinase (BTLA), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), indoleamine 2,3-dioxygenase (IDO) or a combination thereof. In a further aspect, the checkpoint inhibitor interacts with a ligand of a checkpoint protein, including but not limited to CTLA-4, PD-1, B7-H3, B7-H4, TIM3, GAL9, LAG3, VISTA, KIR, BTLA, TIGIT or a combination thereof. In a preferred embodiment, the checkpoint protein inhibitor is an antibody (e.g., a monoclonal antibody, a chimeric antibody, a human antibody or a humanized antibody), antibody fragment or fusion protein that specifically binds to an immune checkpoint protein or a ligand thereof.

In some preferred embodiments, the immune checkpoint inhibitor of the combination is an antibody or antigen-binding fragment thereof that specifically binds to (and inhibits) PD-1, PD-L1, PD-L2, TIGIT, TIM3, LAG3 or CTLA4. As a non-limiting example, a method of treating and/or preventing cancer in a mammal is provided, comprising co-administering an effective amount of (i) a replicative oncolytic vaccinia virus by intratumoral injection and (ii) a CTLA4 and/or PD-1 inhibitor.

In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-1 or PD-L1, preferably selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody that selectively binds to CTLA4, preferably selected from the group consisting of ipilimumab and tremelimumab. In some embodiments, multiple checkpoint inhibitors are administered to the subject in combination with an oncolytic vaccinia virus. In some embodiments, the subject comprises: (a) a CTLA4 inhibitor and a PD-1 inhibitor and a replicating oncolytic vaccinia virus; (b) a CTLA4 inhibitor and an IDO inhibitor and a replicative oncolytic vaccinia virus; (c) a PD-1 inhibitor and an IDO inhibitor and a replicative oncolytic vaccinia virus; or (d) a PD-1 inhibitor and a CTLA4 inhibitor and an IDO inhibitor and a replicative oncolytic vaccinia virus; (e) a LAG3 inhibitor and a PD-1 inhibitor and a replicative oncolytic vaccinia virus; or (f) a TIGIT inhibitor and a PD-1 inhibitor and a replicative oncolytic vaccinia virus are administered in combination. In some embodiments, the replicative oncolytic vaccinia virus is a Wyeth strain, a Western Reserve strain, a Lister strain, or a Copenhagen strain. In some embodiments, the vaccinia virus comprises one or more genetic modifications to increase the selectivity of the virus for cancer cells, preferably the virus is engineered to lack a functional thymidine kinase and/or to lack a functional vaccinia growth factor. In some embodiments, the vaccinia virus comprises functional 14L and/or F4L genes.

In some embodiments, the vaccinia virus is a Wyeth strain, a Western Reserve strain, a Lister strain or a Copenhagen strain having one or more genetic modifications that increase the selectivity of the vaccinia virus for cancer cells, such as inactivation of the thymidine kinase (TK) gene and/or the vaccinia virus growth factor (VGF) gene. In related embodiments, the vaccinia virus is engineered to express a cytokine selected from, for example, but not limited to, GM-CSF, IL-2, IL-4, IL-5 IL-7, IL-12, IL-15, IL-18, IL-21, IL-24, IFN-γ and/or TNF-α, preferably IFN-γ, TNF-α, IL-2, GM-CSF and IL-12. In another related embodiment, the replicative oncolytic vaccinia virus binds to a tumor antigen, such as, but not limited to, BAGE, GAGE-1, GAGE-2, CEA, AIM2, CDK4, BMI1, COX-2, MUM-1, MUC-1, TRP-1 TRP-2, GP100, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3, Nestin, OLIG2, SOX2, Human papillomavirus-E6, Human papillomavirus-E7, ART1, ART4, SART1, SART2, SART3, B-cyclin, β-catenin, Gli1, Cav-1, Cathepsin B, CD74, E-cadherin, EphA2/Eck, Fra-1/Fosl 1, Ganglioside/GD2, GnT-V, β1,6-N, Her2/neu, Ki67, Ku70/80, IL-13Ra2, MAGE-1, MAGE-3, NY-ESO-1, MART-1, PROX1, PSCA, SOX10, SOX11, survivin, caspase-8, UPAR, CA-125, PSA, p185HER2, CD5, IL-2R, Fap-α, tenascin, melanoma-associated antigen p97, WT-1, regulator of G-protein signaling 5 (RGS5), survivin (BIRC5 = baculovirus inhibitor of apoptosis repeat-containing 5), insulin-like growth factor-binding protein 3 (IGF-BP3), thymidylate synthase (TYMS), hypoxia-inducible protein 2, hypoxia-inducible fat droplet-associated (HIG2), matrix metallopeptidase 7 (MMP7), prune homolog 2 (PRUNE2), RecQ protein-like (DNA helicase Q1-like) (RECQL), leptin receptor (LEPR), ERBB receptor feedback inhibitor 1 (ERRFI1), lysosomal protein transmembrane 4 alpha (LAPTM4A); Engineered to express RAB1B, RAS oncogene family (RABIB), CD24, Homo sapiens thymosin beta 4, X-linked (TMSB4X), Homo sapiens S100 calcium binding protein A6 (S100A6), Homo sapiens adenosine A2 receptor (ADORA2B), chromosome 16 open reading frame 61 (C16orf61), ROD1 regulator of differentiation 1 (ROD1), NAD-dependent deacetylase sirtuin-2 (SIR2L), tubulin alpha 1c (TUBA1C), ATPase inhibitor factor 1 (ATPIF1), substrate antigen 2 (STAG2), nuclear casein kinase and cyclin-dependent substrate 1 (NUCKS1). In some embodiments, the tumor antigen is a renal cell carcinoma tumor antigen. In some embodiments, the renal cell carcinoma tumor antigen is regulator of G-protein signaling 5 (RGS5), survivin (BIRC5 = baculovirus inhibitor of apoptosis repeat-containing 5), insulin-like growth factor-binding protein 3 (IGF-BP3), thymidylate synthase (TYMS), hypoxia-inducible protein 2, hypoxia-inducible fat droplet-associated (HIG2), matrix metallopeptidase 7 (MMP7), Prune homolog 2 (PRUNE2), RecQ protein-like (DNA helicase Q1-like) (RECQL), leptin receptor (LEPR), ERBB receptor feedback inhibitor 1 (ERRFI1), lysosomal protein transmembrane 4 alpha (LAPTM4A); selected from the group consisting of RAB1B, RAS oncogene family (RABIB), CD24, Homo sapiens thymosin beta 4, X-linked (TMSB4X), Homo sapiens S100 calcium binding protein A6 (S100A6), Homo sapiens adenosine A2 receptor (ADORA2B), chromosome 16 open reading frame 61 (C16orf61), ROD1 regulator of differentiation 1 (ROD1), NAD-dependent deacetylase sirtuin-2 (SIR2L), tubulin alpha 1c (TUBA1C), ATPase inhibitor factor 1 (ATPIF1), substrate antigen 2 (STAG2), and nuclear casein kinase and cyclin-dependent substrate 1 (NUCKS1).

In some embodiments, the vaccinia virus is administered in an amount of about 10 ⁷ to about 10 ¹¹ pfu, preferably about 10 ⁸ to 10 ¹⁰ pfu, more preferably about 10 ⁹ to 10 ¹⁰ pfu. In some embodiments, the checkpoint inhibitor is administered in an amount of about 2 mg/kg to 15 mg/kg.

In some embodiments, the pharmaceutical combination is administered to a mammal for treating and/or preventing cancer in the mammal. In some embodiments, the cancer is a solid tumor type cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, hepatocellular carcinoma, renal cell carcinoma, bladder cancer, head and neck cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, mesothelioma, gastrointestinal cancer, leukemia, lung cancer (including non-small cell lung cancer), stomach cancer, esophageal cancer, mesothelioma, colorectal cancer, sarcoma, or thyroid cancer. In another preferred embodiment, the pharmaceutical combination is administered to a mammal for treating metastasis. In some embodiments, the subject has renal cell carcinoma.

In a preferred embodiment, the mammal to be treated with the pharmaceutical combination is a human subject. In a related aspect, the subject in need of treatment is a human having a cancer that is refractory (or resistant) to treatment with one or more chemotherapeutic agents and/or refractory to treatment with one or more antibodies. In a specific embodiment, the human has a cancer (e.g., colorectal cancer) that is refractory (or resistant) to treatment comprising an immune checkpoint inhibitor and optionally also refractory to treatment with one or more chemotherapeutic agents. In another embodiment, the human in need of treatment is a human identified as a candidate for therapy with one or more immune checkpoint inhibitors.

In some embodiments, the subject has failed at least one prior chemotherapy or immunotherapy treatment. In some embodiments, the subject has a cancer that is refractory to immune checkpoint inhibitor therapy, preferably the cancer is resistant to treatment with an anti-PD-1 antibody and/or an anti-CTLA-4 antibody. In some embodiments, the subject is identified as a candidate for immune checkpoint inhibitor therapy. In some embodiments, the method comprises administering to the subject an additional therapy selected from chemotherapy (alkylating agents, nucleoside analogs, cytoskeletal modifiers, cytostatic agents) and radiotherapy. In some embodiments, the method comprises administering to the subject an additional anti-cancer virus therapy (e.g., rabies virus, Semliki forest fever virus). In some embodiments, the subject is a human. In some embodiments, the initial dose of replicating anti-cancer vaccinia virus and the initial dose of the immune checkpoint inhibitor are administered to the subject simultaneously, followed by at least one subsequent sequential concurrent administration of the virus and the checkpoint inhibitor to the subject. In some embodiments, the method comprises at least a first, a second, and a third sequential concurrent administration of the replicating oncolytic vaccinia virus and a checkpoint inhibitor to the subject. In some embodiments, the method comprises at least a first, a second, and a third sequential concurrent administration of the replicating oncolytic vaccinia virus and a checkpoint inhibitor to the subject. In some embodiments, the concurrent administration to the subject of a first dose of the replicating oncolytic vaccinia virus and a first dose of the immune checkpoint inhibitor and at least one subsequent sequential concurrent administration of the virus and the checkpoint inhibitor is followed by administration to the subject of at least one dose of the checkpoint inhibitor alone. In some embodiments, the method comprises an interval of from 1 to 3 weeks between the sequential concurrent administrations of the formulations, preferably including an interval of about 1 week, about 2 weeks, or about 3 weeks.

The present application demonstrates that intratumoral administration of a replicating oncolytic vaccinia virus (i) attracts host immune cells (e.g., tumor-infiltrating T cells) to the tumor and (ii) induces expression of several checkpoint proteins, including PD-1, PD-L1, CTLA-4, LAG3, TIM3 and TIGIT, on tumor cells, thereby sensitizing tumor cells to concurrent treatment with inhibitors of the checkpoint protein(s).

Thus, in some embodiments, the expression level of one or more of these gateway proteins is used as a biomarker for selecting human cancer patients for treatment with the combination therapy described herein based on their expression level(s). In some embodiments, expression can be measured using any assay for measuring protein levels. In some embodiments, protein expression can be measured using an assay, such as FACS or Nanotring assay.

In a related embodiment, the human in need of treatment is a human having a tumor that does not express a checkpoint protein (e.g., a checkpoint inhibitor refractory subject) or expresses a checkpoint protein at relatively low levels, wherein the oncolytic vaccinia virus component of the combination therapy is administered in an amount effective to sensitize the tumor to an immune checkpoint inhibitor of the combination by inducing expression of the checkpoint protein (e.g., PD-L1). In some embodiments, the human can have a tumor that does not express PD-1, PD-L1, CTLA-4, LAG3, TIM3, and/or TIGIT, or that expresses one or more of these checkpoint proteins at relatively low levels, and the oncolytic vaccinia virus is administered in an amount effective to sensitize the tumor to a PD-1, PD-L1, CTLA-4, LAG3, TIM3, and/or TIGIT inhibitor. In another related embodiment, the level of the checkpoint protein is measured in the tumor prior to administration of the combination therapy of the anti-cancer vaccinia virus and the checkpoint inhibitor, and if it is determined that the checkpoint protein is not expressed or is expressed at relatively low levels in the tumor, the combination therapy is administered to the subject. In another related embodiment, a method is provided for sensitizing a tumor to a checkpoint inhibitor, comprising administering to a human having the tumor an amount of an anti-cancer vaccinia virus effective to induce expression of the checkpoint protein in the tumor, and co-administering to the human a checkpoint inhibitor. In some aspects, a tumor that does not express the checkpoint protein or expresses the checkpoint protein at relatively low levels means that less than 50%, less than 25%, less than 15%, less than 10%, less than 5%, less than 1%, or less than 0.5% of the tumor cells stain positive for the checkpoint protein as assessed by immunohistochemistry (IHC) of a tumor sample. See, for example, IIie et al., Virchows Arch, 468(5):511-525 (2016). In some embodiments, the human has non-small cell lung cancer, stomach cancer, renal cell carcinoma, pancreatic cancer, or colorectal cancer.

In another related embodiment, the human in need of treatment is a human having an immunologically "cold" tumor (meaning that the tumor is substantially or relatively devoid of immune cells in the tumor microenvironment). Treatment with the oncolytic vaccinia virus attracts immune cells (e.g., T cells) into the tumor and acts synergistically with checkpoint inhibitors to treat the tumor. "Cold" tumors can be identified by methods known in the art, including but not limited to, single staining or multiplex immunohistochemistry (IHC) for immune markers such as CD3 and CD8 in the tumor center and infiltrating region, flow cytometry for phenotype, genomic analysis of tumor tissue, RNA profiling of tumor tissue, and/or cytokine profiling in serum.

The anticancer vaccinia virus and the immune checkpoint inhibitor of the combination are administered in parallel (e.g. simultaneously) and may be administered as parts of the same formulation or in different formulations. By simultaneous (or parallel) administration it is meant that the initial doses of each of the combination partners are administered simultaneously or approximately simultaneously (within 24 hours of each other, preferably within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 hours of each other or within 1 hour of each other), and preferably at least one subsequent dose of each of the combination partners is administered simultaneously or approximately simultaneously. Thus, in one aspect, the combination therapy as described herein comprises an initial dose of a replicating oncolytic vaccinia virus administered concurrently with an initial dose of a checkpoint inhibitor (e.g., treatment of a subject with the combination therapy involves at least a first administration, wherein the oncolytic vaccinia virus and the checkpoint inhibitor are administered to the subject concurrently), and preferably further comprises at least 1, 2, 3, 4, or more additional sequential concurrent administrations of the oncolytic vaccinia virus and the checkpoint inhibitor. Thus, the combination treatment regimen with the pharmaceutical combination can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or more sequential concurrently administered doses of the agent. In preferred embodiments, the interval between consecutive co-administered doses of the anti-cancer vaccinia virus and the checkpoint inhibitor is in the range of from about 1 day to about 3 weeks or any interval therebetween, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days. In some preferred embodiments, the interval between consecutive co-administered doses of the anti-cancer vaccinia virus and the checkpoint inhibitor is about 1 week or about 2 weeks. Following at least one initial co-administered dose of the anti-cancer virus and the checkpoint inhibitor, one or more doses of the checkpoint inhibitor alone can be administered to the subject.

In another aspect, the present invention provides a commercial package comprising a combination of an anti-cancer vaccinia virus as described herein and an immune checkpoint inhibitor as active agents, together with instructions for concurrent use in therapy and/or prevention as described herein. In a preferred aspect, the commercial package comprises a combination of a Western Reserve, Copenhagen, Wyeth or Lister strain vaccinia virus as active agents and a PD-1, PD-L1, TIGIT or CTLA4 inhibitor.

In some embodiments, the invention provides a method of treating a tumor in a human, comprising co-administering to the human a combination comprising (a) a replicating oncolytic vaccinia virus and (b) an inhibitor of an immune checkpoint protein. In some embodiments, the replicating oncolytic virus is administered intratumorally. In some embodiments, the replicating oncolytic virus is administered intravenously. In some embodiments, the replicating oncolytic virus is administered intraarterially. In some embodiments, the replicating oncolytic virus is administered intraperitoneally. In some embodiments, the replicating oncolytic virus is delivered exclusively via intratumoral administration. In some embodiments, the replicating oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraarterially and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic vaccinia virus is administered in an amount effective to induce expression of an immune checkpoint protein in the tumor. In some embodiments of the method of treatment, the immune checkpoint protein is selected from PD-1, PD-L1, CTLA-4, LAG3, TIM3, and TIGIT. In some embodiments, the invention provides a method of treating a tumor in a human, comprising co-administering to the human a combination comprising (a) a replicating oncolytic vaccinia virus in an amount effective to induce expression of an immune checkpoint protein in the tumor, and (b) an inhibitor of an immune checkpoint protein. In some embodiments, the replicating oncolytic vaccinia virus is administered intratumorally. In some embodiments, the replicating oncolytic vaccinia virus is administered IV. In some embodiments of the method of treatment, the immune checkpoint protein is selected from PD-1, PD-L1, CTLA-4, LAG3, TIM3, and TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is CTLA-4. In some embodiments of the method of treatment, the immune checkpoint protein is PD-L1. In some embodiments of the method of treatment, the immune checkpoint protein is LAG3. In some embodiments of the method of treatment, the immune checkpoint protein is TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is PD-1. In some embodiments of the method of treatment, the immune checkpoint protein is TIM3. In some embodiments of the method of treatment, the tumor is a solid tumor. In some embodiments of the method of treatment, the tumor is colorectal cancer. In some embodiments of the method of treatment, the tumor is renal cell carcinoma.

In some embodiments of the dual combination therapy method, the inhibitor of the immune checkpoint protein is a monoclonal antibody that selectively binds to PD-1 or PD-L1. In some embodiments, the monoclonal antibody that selectively binds to PD-1 or PD-L1 is selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab.

In some embodiments of the dual combination therapy method, the inhibitor of the immune checkpoint protein is a monoclonal antibody that selectively binds to CTLA-4. In some embodiments, the monoclonal antibody that selectively binds to CTLA-4 is selected from the group consisting of ipilimumab and tremelimumab.

In some embodiments of the dual combination therapy, the tumor does not express an immune checkpoint protein or expresses a relatively low level of an immune checkpoint protein prior to administration of the replicating oncolytic vaccinia virus.

In some embodiments of the dual combination therapy method, the method comprises a step of measuring the level of expression of an immune checkpoint protein in the tumor prior to administering the combination.

In some embodiments, the invention provides a method of treating a tumor in a human, comprising co-administering to the human a combination comprising (a) a replicating oncolytic vaccinia virus, (b) an inhibitor of PD-1 and/or PD-L1, and (c) an inhibitor of an immune checkpoint protein. In some embodiments, the replicating oncolytic vaccinia virus is administered in an amount effective to induce expression of an immune checkpoint protein. In some embodiments, the replicating oncolytic virus is administered intratumorally. In some embodiments, the replicating oncolytic virus is administered intravenously. In some embodiments, the replicating oncolytic virus is administered intraarterially. In some embodiments, the replicating oncolytic virus is administered intraperitoneally. In some embodiments, the replicating oncolytic virus is delivered exclusively via intratumoral administration. In some embodiments, the replicating oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraarterially and the checkpoint inhibitor is administered systemically. In some embodiments, the invention provides a method of treating a tumor in a human, comprising co-administering to the human a combination comprising (a) a replicating oncolytic vaccinia virus in an amount effective to induce expression of an immune checkpoint protein in the tumor, (b) an inhibitor of PD-1 and/or PD-L1, and (c) an inhibitor of an immune checkpoint protein, wherein the replicating oncolytic vaccinia virus is administered intratumorally. In some embodiments of the method of treatment, the immune checkpoint protein is selected from CTLA-4, LAG3, TIM3, and TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is CTLA-4. In some embodiments of the method of treatment, the immune checkpoint protein is LAG3. In some embodiments of the method of treatment, the immune checkpoint protein is TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is TIM3. In some embodiments of the method of treatment, the tumor is a solid tumor. In some embodiments of the method of treatment, the tumor is colorectal cancer. In some embodiments of the method of treatment, the tumor is renal cell carcinoma.

In some embodiments of the triple combination therapy method, the inhibitor of the immune checkpoint protein is a monoclonal antibody that selectively binds to PD-1 or PD-L1. In some embodiments, the monoclonal antibody that selectively binds to PD-1 or PD-L1 is selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab.

In some embodiments of the triple combination therapy method, the inhibitor of the immune checkpoint protein is a monoclonal antibody that selectively binds to CTLA-4. In some embodiments, the monoclonal antibody that selectively binds to CTLA-4 is selected from the group consisting of ipilimumab and tremelimumab.

In some embodiments of the triple combination therapy, the tumor does not express an immune checkpoint protein or expresses a relatively low level of an immune checkpoint protein prior to administration of the replicating oncolytic vaccinia virus.

In some embodiments of the triple combination therapy method, the method comprises a step of measuring the level of expression of a checkpoint protein in the tumor prior to administering the combination.

Other embodiments of the present invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the present invention also applies to other aspects of the present invention and vice versa. It is understood that embodiments in the Examples section are embodiments of the present invention that are applicable to all aspects of the present invention.

The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Figures 1A-1C. Figure 1A: Chart depicting the concurrent combination therapy regimen of intratumoral (IT) injection of mJX-594 and intraperitoneally administered anti-PD-1 checkpoint inhibitor antibody. Eight-week-old BALB/c immunocompetent mice were injected with 5×10 ⁵ Renca (renal cancer) cells. Once tumor size reached ≥50㎣, mice were treated with PBS (control, days 0, 3, 6, and 9), anti-PD-1 antibody alone (days 0, 3, 6, and 9), mJX-594 alone (days 0, 2, and 4), or anti-PD-1 and mJX-594 (day 0), delivered in parallel (simultaneous administration of agents on days 0, 2, and 4), followed by intratumoral (IT) administration of 1 × 10 ^{7 pfu of anti-PD-1 alone and mJX-594 and intraperitoneal (IP) administration of 10 mg/kg of anti-PD-1 on day 6. Figure 1B: RENCA cells (2 × 10 6 cells} ) in 100 ㎕ of PBS were injected subcapsularly of the left kidney into 8-week-old female BALB/c mice. At day 10 post-transplant, mice bearing Renca tumors (50 mm3 to 100 mm3 as visualized by IVIS® Spectrum in an in vivo imaging system) were intraperitoneally (ip) administered (i) PBS (control), (ii) vaccinia virus (JX-929) monotherapy (6×10 ⁷ PFU on days 10, 11, and 12 post-transplant for a total of three doses), (iii) anti-PD1 monotherapy (BioXcell, West Lebanon, NH; 100 μl) on days 10, 11, and 12 post-transplant for a total of three doses), or (iv) concurrent JX929 plus anti-PD1 treatment (administered on days 10, 11, and 12 post-transplant, respectively, with JX-929 administered in the morning and ICI administered 9 hours apart in the afternoon of the same day). Treatment was performed according to the indicated regimen. Figure 1C: Balb/c mice bearing Renca tumors exceeding 50 mm3 were administered four intratumoral doses of mJX594 (1 × 10 ⁷ on days 0, 3, 6, and 9, respectively) or PBS control according to the indicated treatment regimen.
FIG. 2A-2B. FIG. 2A: Graph depicting the effect of the treatment regimens described in FIG. 1A on tumor volume. The concurrent combination treatment (PD1+mJX594) significantly inhibited tumor growth (18 days post-implantation) compared to all other treatment groups. FIG. 2B: Photograph and graph depicting tumor weights in each treatment group described in FIG. 1A. The concurrent combination treatment (PD1+mJX594) synergized to significantly reduce tumor volume compared to either monotherapy alone.
FIG. 3A-3B. Drawings showing that concurrent combination therapy with IT mJX-594 and anti-PD-1 significantly increases intratumoral T-cell infiltration compared to control and monotherapy with either agent. Mice were treated according to the dosing regimen depicted in FIG. 1 . FIG. 3A: Images showing a significant increase in CD8 T-cell infiltration in both peritumoral and intratumoral regions in the concurrent combination therapy group compared to the control and monotherapy groups. FIG. 3B: Graph showing a significant increase in peritumoral and intratumoral CD8 T-cell infiltration in the concurrent combination therapy group compared to the control and monotherapy either agent.
FIG. 4A-4B. Drawings showing that concurrent combination therapy with IT mJX-594 and anti-PD-1 upregulates intratumoral PD-L1 expression. Mice were treated according to the dosing regimens depicted in FIG. 1. FIG. 4A: Images showing a significant increase in PD-L1 expression levels in both peritumoral and tumor core regions in the concurrent combination therapy group compared to the control and monotherapy groups alone (PD-L1 staining). FIG. 4B: Images showing a significant increase in intratumoral apoptosis in the concurrent combination therapy group compared to the control and monotherapy groups.
Figures 5a-5b. Drawings showing that CD8 T cells and CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) are increased in the concurrent combination therapy group compared to the control group. Figure 5a: Flow cytometry graph showing positivity for CD8 and Gr-1 in tumors from each group, showing a significant increase in CD8+ T cells for tumors from the concurrent combination group compared to the monotherapy. An increase in MDSC is also shown for one of the monotherapy groups. Figure 5b: Bar graph showing the results of flow cytometry.
Chart illustrating the combination therapy regimen of IT injections of mJX-594 and anti-PD1 (+/- anti-CTLA4) checkpoint inhibitor antibodies delivered intraperitoneally. 5 × 10 ⁵ Renca cells were injected subcutaneously into the right flank of 8-week-old BALB/c mice. Treatment was initiated (day 0) when tumors reached 50-100 mm 3 in size. On day 0, mice (bearing Renca tumors) were treated with PBS (control), the combination of mJX-594+anti-PD1 delivered sequentially, the combination of mJX-594+anti-PD-1 delivered in parallel, and the triple combination of mJX-594+anti-PD-1+anti-CTLA4 delivered in parallel. mJX-594 was administered at 1× ¹⁰⁷ pfu IT, anti-PD1 at 10 mg/kg IP, and anti-CTLA4 at 4 mg/kg IP.
Figure 7. A graph illustrating the effect of the treatment regimens described in Figure 6 on tumor volume. The concurrent combination treatment with αPD1+mJX594 and αPD1+mJX594+αCTLA4 significantly inhibited tumor growth from day 6 (post-treatment) compared to all other treatment groups, and both concurrent combination treatment groups significantly delayed tumor growth compared to the control and sequential combination treatment groups (mJX594->αPD1), with tumor regression observed from day 12.
FIG. 8. Chart depicting the combination therapy regimen with IT injections of mJX-594 and anti-CTLA4 checkpoint inhibitor antibody delivered intraperitoneally. 5 × 10 ⁵ Renca cells were injected subcutaneously into the right flank of 8-week-old BALB/c mice. When tumors reached 50-100 mm3 in size, treatment was initiated (day 0). On day 0, mice (bearing Renca tumors) were treated with PBS (control), anti-CTLA4 alone, mJX-594 alone, the combination of mJX-594+CTLA4 delivered sequentially, and the combination of mJX- 594+CTLA4 delivered in parallel. mJX-594 was administered at 1 × 10 ⁷ pfu IT and anti-CTLA4 at 4 mg/kg.
Figure 9. A graph depicting the effect of the treatment regimens described in Figure 8 on tumor volume. Concurrent combination therapy with mJX594+αCTLA4 significantly delayed tumor growth compared to sequential combination therapy with mJX594+αCTLA4 or either monotherapy alone.
Figures 10a-10b. Drawings showing that the parallel combination of IT mJX594 and anti-CTLA4 significantly increases CD8+ T-cell tumor infiltration and decreases MDSC levels compared to either the sequential combination or monotherapy alone. Figure 10a: Flow cytometry graphs showing positivity for CD8 and Gr-1 in tumors from each treatment group, demonstrating a significant increase in CD8+ T-cells and a significant decrease in MDSC for tumors from the parallel combination group compared to the sequential treatment group or either monotherapy alone. Figure 10b: Bar graph illustrating the flow cytometry results.
Figure 11. Chart depicting combination therapy regimens with intravenous (IV) injection of mJX-594 and intraperitoneally delivered anti-PD1 checkpoint inhibitor antibody. 5 × 10 ⁵ Renca cells were injected subcutaneously into the right flank of 8-week-old BALB/c mice. Treatment was initiated (day 0) when tumors reached 50-100 mm3 in size. On day 0, mice (bearing Renca tumors) were treated with PBS (control), anti-PD1 alone, mJX-594 alone, or mJX-594+anti-PD1 delivered in parallel. mJX-594 was administered at 2 × 10 ⁷ pfu IV and anti-PD1 was administered at 10 mg/kg IP.
Figure 12. Graph depicting the effect of the treatment regimen described in Figure 11 on tumor volume. Concurrent combination treatment with mJX594 IV+αPD1 was inferior to treatment with mJX594 alone and no better than treatment with αPD1 alone.
Figure 13. A chart showing the 4-fold change (relative to pretreatment levels) in immune checkpoint proteins in Renca tumor-bearing mice treated intratumorally with four 1×10 ⁷ pfu doses of mJX594 (Wyeth vaccinia virus engineered to contain a disruption of the viral thymidine kinase gene and an insertion of murine GM-CSF) administered every 3 days.
Figure 14. Schematic providing data on the number of mJX594 injections and tumor growth inhibition. To find the optimal immunotherapy using mJX594, various doses were tested in Renca renal cell carcinoma. Tumor growth was reduced as the number of mJX594 doses increased.
Figure 15. Image depiction showing intratumoral recruitment of CD8+ T cells following mJX594 treatment.
Figure 16. Image depiction of intratumoral recruitment of CD8+ T cells following mJX594 treatment. In mJX594-treated tumors, aggregates of CD8+ lymphoid cells resembling lymphoid follicles were observed.
Figure 17. Data showing that mJX594 increases the number and effector function of intratumoral CD8+ T cells. The ratio of CD8+ T cells to regulatory T cells was increased after mJX594 treatment. The expression of ICOS and granzyme B in CD8+ T cells was increased after mJX594 treatment. The number of CD8+ and CD4+ T cells as well as CD4+Foxp3+CD25+ regulatory T cells were simultaneously expanded in the lymphoid cell compartment, but the ratio of CD8+ effector T cells to regulatory T cells was further increased compared to the control. Additionally, the expression of ICOS and granzyme B (GzB), which are costimulatory and activation markers of T cells, was increased in CD8+ T cells.
Figure 18. Data showing that mJX594 treatment repolarizes myeloid cells (Ly6G-Ly6C+↑, Ly6G+Ly6Cint↓). mJX594 increases CD11b+Ly6G-Ly6C+ monocytic myeloid cells and decreases CD11b+Ly6G+Ly6Cint granulocytic myeloid cells. In subpopulation analysis of the myeloid cell compartment, we found an increase in CD11b+Ly6G-Ly6C+ monocytic myeloid cells and a decrease in granulocytic myeloid cells by CD11b+Ly6G+Ly6Cint, indicating a significant increase in the monocyte to granulocyte ratio by mJX594 administration.
Figure 19. Schematic diagram showing data for treatment by depletion antibody experiments. To understand which components of the immune system are responsible for the therapeutic efficacy following mJX594 treatment, the effects of depletion on CD8+ T-cells, CD4+ T-cells, and GM-CSF on tumor growth and antitumor immunity were tested.
Figure 20. Data showing that depletion of T cells or GM-CSF significantly abolished the antitumor effect of mJX594. Both CD8+ and CD4+ T cells are essential mediators of the antitumor effect of mJX594 treatment, and GM-CSF could also provide immunotherapeutic benefit. Effective tumor inhibition was detected with mJX594 monotherapy, but depletion of either CD8+ or CD4+ T cells resulted in abrogation of the therapeutic effect.
Figure 21. Data showing that depletion of CD4+ T-cells or GM-CSF attenuated intratumoral CD8+ T-cell infiltration after mJX594. Depletion of CD4+ T-cells decreased intratumoral CD8+ T-cells, suggesting that CD4+ T-cells are involved in the activation of CD8+ T-cells. Depletion of GM-CSF decreased both CD8+ and CD4+ T-cells. Depletion of CD4+ T-cells by mJX594 injection decreased intratumoral CD8+ T-cells, suggesting that CD4+ T-cells are involved in the activation of CD8+ T-cells. In contrast, depletion of CD8+ T-cells did not induce significant changes in CD4+ T-cells, indicating that CD8+ T-cells do not affect CD4+ T-cells. These data indicate that treatment with mJX594 induced CD8+ and CD4+ T-cell priming, which is indicative of antitumor immunity. Infiltration of CD8+ and CD4+ T-cells is indicative of antitumor effects.
Figure 22. Schematic diagram of the experiment for the triple combination therapy of mJX594, αPD-1, and αCTLA-4.
FIG. 23. Data showing that the triple combination of mJX594, αPD-1 and αCTLA-4 significantly delayed tumor growth. In particular, the triple combination of mJX594, αPD-1 and αCTLA-4 caused complete regression of Renca tumors in some mice (37.5%). The dual combination of αPD-1 and αCTLA-4 delayed tumor growth by 14.5% and mJX594 monotherapy inhibited tumor growth by 36.9% compared to the control, whereas the triple combination showed 76.5% tumor growth inhibition. In particular, the triple combination of mJX594, αPD-1 and αCTLA-4 caused complete tumor regression (complete response rate: 37.5%) that was not observed in tumors treated with either the dual combination or mJX594 monotherapy.
Figure 24. Data showing that triple combination immunotherapy of mJX594, PD-1 and CTLA-4 extends overall survival. Mice treated with the triple combination therapy exhibited remarkable anticancer therapeutic effects. Furthermore, to determine whether these strong anticancer effects induced by the triple combination therapy could translate into long-term survival benefits, survival analyses of tumor-bearing mice were performed. Mice treated with the triple combination therapy exhibited survival benefits compared to either monotherapy or dual combination immunotherapy.
Figure 25. Schematic diagram illustrating the experimental setup for the triple combination therapy of mJX594, αPD-1, and αLAG3.
Figure 26. Data showing that the triple combination of mJX594, αPD-1 and αLAG3 moderately delayed tumor growth. In this experiment, the triple combination did not show statistically significant differences compared to the dual combination of mJX594 and αPD-1. The dual combination of mJX594 and αPD-1 delayed tumor growth by 41.9% compared to the control, whereas αLAG3 monotherapy inhibited tumor growth by 5.7%. The triple combination showed tumor growth inhibition of 30.1%.
Figure 27. Data showing that the triple combination of mJX594, αPD-1 and αLAG3 increased CD8+ and CD4+ T-cells. Subpopulation analysis of the lymphoid cell compartment showed an increase in absolute numbers of intratumoral CD8+ and CD4+ T-cells with the dual and triple combination treatment.
Figure 28. Schematic diagram illustrating the experimental setup for the triple combination therapy of mJX594, αPD-1, and αTIGIT.
Figure 29. Data showing that the triple combination of mJX594, αPD-1, and αTIGIT modestly delays tumor growth. The triple combination did not show significant differences compared to the dual combination of mJX594 and αPD-1 in Renca tumors.
Figure 30. Data showing that the triple combination of mJX594, αPD-1 and αTIGIT increased CD8+ and CD4+ T-cells. Subpopulation analysis of the lymphoid cell compartment showed an increase in the absolute number of intratumoral CD8+ and CD4+ T-cells with the dual and triple combination treatment.
Figure 31. Schematic showing data showing that mJX594 synergizes with anti-PD1 therapy to delay colon cancer growth. To overcome resistance to ICI monotherapy, the combination efficacy of mJX594 and immune checkpoint blockade was evaluated in the CT26 colon cancer model. In this model, αPD-1 monotherapy had little effect on tumor growth, with mJX594 monotherapy modestly inhibiting tumor growth. However, the combination of mJX594 and αPD-1 antibody dual therapy significantly delayed tumor growth.
Figure 32. Data showing that the combination of mJX594 and anti-PD1 therapy increased intratumoral CD8+ T cells. In accordance with tumor growth inhibition, microscopic analysis showed a notable recruitment of CD8+ T cells in both the peritumoral and tumor core areas treated with the combination therapy.
Figures 33A-G. Schematic showing that mJX594 (JX) induces dynamic changes in immune-related genes in the immunosuppressive TME. Renca tumors were implanted sc into BALB/c mice and treated with a single i.t. injection of 1 × 10 ⁷ pfu of mJX-594 when tumors reached >50 mm3. (A) Representative image of Renca tumors treated with JX. Tumors were stained for vaccinia virus (VV), CD31, CD8, CD11c, and PD-L1. (B) Quantification of vaccinia virus ⁺ , CD31 ⁺ blood vessels, CD8 ⁺ cytotoxic T cells, CD11c ⁺ dendritic cells, and PD-L1 ⁺ cell expression. (C) Temporal changes in VV, CD8, and PD-L1 in the tumor microenvironment following JX treatment. (D) Images showing upregulated PD-L1 expression (red) in various cell types (green) within the TME following JX treatment. Note that PD-L1 expression was predominantly observed in Pan-CK ⁺ tumor cells (arrowheads), but some CD11b ⁺ myeloid cells (arrowheads) also occasionally expressed PD-L1, whereas CD3 ⁺ T cells did not. (E) Nanostring immune-related gene expression heatmap. Red and green indicate up- and downregulation, respectively. (F) Volcano plot showing gene expression in JX-treated tumors. Genes associated with immune stimulation are shown. Red indicates p < 0.05. (G) Comparison of gene expression associated with inhibitory immune checkpoint (IC), agonistic IC, Th1 response, Th2 response, TME, and myeloid cells. n = 5 for each group unless otherwise specified. Values are mean ± SEM. *p < 0.05 vs. control. Scale bar, 50 μm. Some data is also shown in Fig. 13.
FIGS. 34A-34M. JX inhibits tumor growth by increased T cell infiltration and modulation of myeloid cells. Renca tumor-bearing mice were treated 1-3 times i.t. with PBS or 1×10 ⁷ pfu of JX. (A-B) Comparison of tumor growth in mice treated with JX. Mean (A) and individual (B) tumor growth curves over time. (C-D) Representative images (C) and comparison (D) of CD8 ⁺ T cells in peritumoral or intratumoral regions of tumors treated 1-3 times with JX. (E) Representative flow cytometry plots showing intratumoral CD8 ⁺ and CD4 ⁺ T cell fractions. (F) Absolute numbers of CD8 ⁺ and CD4 ⁺ T cells per gram of tumor as calculated from flow cytometry. (G) Comparison of CD45 ⁺ , CD8 ⁺ , and CD4 ⁺ fractions in tumors treated with triple doses of JX. (H-J) Comparison of the fractions of CD4 ⁺ Foxp3 ⁺ CD25 ⁺ (Treg), CD8/Treg ratio, CD8 ⁺ ICOS ⁺ , and CD8 ⁺ GzB ⁺ in tumors. (K) Comparison of the fractions of CD11b ⁺ Gr1 ⁺ myeloid cells in tumors. (L) Representative flow cytometry plots showing the fraction of CD11b ⁺ myeloid cells in tumors. (M) Comparison of the fractions of Ly6G ^- Ly6C ⁺ monocytic myeloid cells, Ly6G+Ly6C ^int granulocytic myeloid cells, and monocyte/granulocyte ratio relative to CD11b+ in tumors. Unless otherwise specified, n = 5 to 6 for each group. Values are means ± SEM. ^* p < 0.05 vs. control; ^# p < 0.05 vs. JX x1; ^$ p < 0.05 vs. JX x2. ns, not significant. Scale bars, 100 μm. Some data are also shown in Figures 19 to 21.
Figures 35A-35E. Intratumoral injection of JX induced CD8 ⁺ lymphocyte infiltration in both local and distant tumors. Mice were injected sc with Renca in the right flank and with Renca or CT26 tumors in the left flank. Arrows indicate it JX treatment. (A) Schematic diagram of tumor grafting and treatment, and growth curves for JX-injected Renca tumors and non-injected Renca tumors. (B-C) Representative images (B) and comparison (C) of CD8 ⁺ T cells (green) in JX-injected and non-injected tumors. (D) Schematic diagram of grafting and treatment, and growth curves for JX-injected Renca tumors and non-injected CT26 tumors. (E-F) Representative images (E) and comparison (F) of CD8 ⁺ T cells (green) in JX-injected tumors and distant tumors. Unless otherwise specified, n = 5 for each group. Values are mean ± SEM. *p < 0.05, ns, not significant vs. control. Scale bar, 50 μm.
FIGS. 36A-36D. Anti-tumor immunity plays a critical role in the overall therapeutic efficacy of JX. Mice were transplanted sc with Renca and then treated with it JX or i.p. depleting antibodies against CD8 ⁺ , CD4 ⁺ T cells or mouse GM-CSF. (A) Treatment schematic. (B-C) Comparison of tumor growth in mice treated with JX or depleting antibodies. Mean (B) and individual (C) tumor growth curves over time. ^* p < 0.05 vs. control; ^$ p < 0.05 vs. αGM-CSF. (D-E) Absolute numbers of CD8 ⁺ (D) and CD4 ⁺ (E) T cells per gram of tumor treated with JX and immune cell-depleting antibodies. n = 7-8 for each group unless otherwise specified. Values are means ± SEM. ^* p < 0.05 vs. control; ^# p < 0.05 vs. VX; For αGM-CSF, ^$ p < 0.05. ns, not significant.
FIGS. 37A-37E. Combination therapy of JX and αPD-1 synergistically induces CD8 ⁺ T cell-mediated tumor immunity. Renca tumor-bearing mice were treated with either PBS, JX, αPD-1 antibody, or JX plus αPD-1 antibody. (A-B) Comparison of tumor growth in mice treated with JX and/or αPD-1 antibody. Mean (A) and individual (B) tumor growth curves over time. (C-D) Representative images (C) and comparison (D) of CD8 ⁺ T cells, CD31 ⁺ blood vessels, activated caspase 3 (Casp3) ⁺ apoptotic cells, and PD-L1 ⁺ cells in tumors treated with JX and/or αPD-1 antibody. (E) Diagram depicting overcoming of the immunosuppressive TME by combination therapy of JX and αPD-1 blockade. n = 7 for each group unless otherwise specified. Values are means ± SEM. *p < 0.05 for control; ^#p < 0.05 for JX; ^$ p < 0.05 for αPD-1. ns, not significant. Scale bar, 100 μm. Some data are also shown in Figures 19-21.
Figures 38A-38F. The efficacy of combination immunotherapy with intratumoral JX and systemic ICI is not significantly affected by treatment schedule. Mice were implanted sc with Renca tumors and treated with JX + ICI on various schedules. (A) Diagram depicting various treatment schedules. Arrows indicate treatment with either it delivery of JX (red arrows) or systemic delivery of immune checkpoint blockade (blue arrows). (B-C) Comparison of tumor growth in mice treated with JX and αPD-1 antibody using different treatment schedules. Mean (B) and individual (C) tumor growth curves over time. (D) Representative flow cytometry plots showing tumor-infiltrating CD8 ⁺ and CD4 ⁺ T cell fractions. (E-F) Comparison of absolute numbers of CD8 ⁺ , CD4 ⁺ , CD8 ⁺ ICOS ⁺ , and CD8 ⁺ GzB ⁺ cells per gram of tumor. Unless otherwise specified, n = 7 for each group. Values are means ± SEM. *p < 0.05, ns, not significant vs. control.
FIG. 39A-39E. Triple combination of JX, αPD-1, and αCTLA-4 antibodies results in complete regression and improved overall survival. Mice were implanted sc with Renca tumors and treated with JX in the presence or absence of immune checkpoint blockade for PD-1 and CTLA-4. (A-B) Comparison of tumor growth in mice treated with JX and/or immune checkpoint blockade. Mean (A) and individual (B) tumor growth curves over time. (C) Waterfall plot showing the maximum percent change from baseline in tumor size. (D) Kaplan-Meier plot for overall survival. (E) Comparison of tumor sizes after rechallenge with Renca tumor cells in mice with complete tumor regression. n = 8 for each group unless otherwise specified. ^* p < 0.05 vs. control; ^# p < 0.05 vs. JX; ^$ p < 0.05 for αPD-1+ αCTLA-4. ns, not significant. Some data are also shown in Figs. 23 and 24.
Figures 40A-40L. Triple combination therapy delays tumor growth and metastasis in a spontaneous breast cancer model. Tumor growth was analyzed weekly starting at 9 weeks postnatally in spontaneous mammary tumors of MMTV-PyMT mice. Samples were collected at 13 weeks postnatally. (A) Diagram showing the treatment schedule. Arrows indicate treatment with either it delivery of JX or systemic delivery of αPD-1 and αCTLA-4 antibodies. (B) Representative images showing gross appearance of tumors. Dotted circles mark the boundaries of palpable mammary tumor nodules. (C) Comparison of total tumor burden, where tumor burden was calculated by summing the volumes of all tumor nodules per mouse. (D) Comparison of the number of palpable tumor nodules. (E) Comparison of the volumes of individual tumor nodules. Individual tumor nodules in MMTV-PyMT mice are plotted as individual points. (F) Kaplan-Meier curves for overall survival. (G) Tumor sections by H&E showing intratumoral areas. The acinar structures in the JX and JX+P+C groups are early, less invasive lesions (Ea) that have distinct borders from the surrounding breast adipose tissue (Adi). In contrast, the invasive ductal carcinoma areas (Ca) in Cont and P+C massively invaded the surrounding tissue and formed solid sheets of tumor cells without residual acinar structures. Scale bar, 200 μm. (H-J) Representative images and comparison of CD8 ⁺ T cells (H and I) and CD31 ⁺ tumor blood vessels (H and J) in the tumors. (K) Representative lung sections stained with H&E. Arrows indicate metastatic lesions. Scale bar, 200 μm. (L) Comparison of the number of metastatic colonies per lung section. n = 6 to 7 for each group unless otherwise specified. Values are means ± SEM. ^* p < 0.05 vs. control; ^# p < 0.05 for JX; ^$ p < 0.05 for αPD-1+ αCTLA-4. ns, not significant. Scale bar, 100 μm.
Figure 41. Vaccinia virus is not detected in distal tumors. On the right, strong vaccinia virus (VV) replication (green) is observed in the injected tumor, whereas on the left, no vaccinia virus is detected in the non-injected tumor. Scale bar, 100 μm.
FIG. 42A-H. Combination therapy with JX and ^Λ CTLA-4 synergistically induced CD8+ T cell-mediated tumor immunity. Mice bearing Renca tumors were treated with either PBS, JX, αCTLA-4 antibody, or JX plus αCTLA-4 antibody. (A-B) Comparison of tumor growth in mice treated with JX and/or αCTLA-4 antibody. Mean (A) and individual (B) tumor growth curves over time. (C-E) Images and comparison of CD8+ T cells (C and D) and CD31+ blood vessels (C and E) in tumors. (F-H) Absolute numbers of CD45+ immune cells (F), CD8+ T cells (G), and CD4+ cells (H) per gram of tumor challenged with JX and/or αCTLA-4 antibody. Values are means ± SEM. *p < 0.05 vs. control; #p < 0.05 vs. JX; For αCTLA-4, $p < 0.05. ns, not significant. Scale bar, 100 μm.
FIG. 43A-43E. JX enhances antitumor efficacy by ^Λ CTLA-4 immune response independent of treatment schedule. (A-B) Comparison of tumor growth in mice treated with JX and αCTLA-4 antibody using different time schedules. Mean (A) and individual (B) tumor growth curves over time. (C) Representative flow cytometry plots showing tumor-infiltrating CD8+ and CD4+ T cell fractions in tumors. (D-E) Absolute numbers of CD8+, CD4+, CD8+ICOS+, and CD8+GzB+ cells per gram of tumor treated with JX and αCTLA-4 antibody. Values are means ± SEM. *p < 0.05 vs. control, ns, not significant. Some data are also shown in FIG. 38 .

I. Select definition

The terms “inhibiting,” “reducing,” or “preventing,” or any variation of these terms, when used in the claims and/or in this specification, include any measurable reduction or complete inhibition of achieving a desired result.

The term "combination" as used herein means the combined administration of an anticancer agent, i.e., an anticancer vaccinia virus and an immune checkpoint inhibitor, which may be administered independently or by use of fixed combinations of different amounts of combination states. The term "combination" also defines a "kit" comprising combination states to be administered simultaneously. Preferably, the time interval between the successive simultaneous administrations of the combination partners is selected so that the combination of agents exhibits a synergistic effect. The term "synergistic" or "synergistic effect" as used herein means that the effect achieved by the combination of anticancer agents included in the present invention is greater than the sum of the effects resulting from using the anticancer agents, i.e., the anticancer vaccinia virus and the immune checkpoint inhibitor as monotherapy. Advantageously, such synergy provides greater efficacy at the same dose and/or prevents or delays the formation of multidrug resistance.

The term "refractory cancer" as used herein refers to a cancer that does not respond favorably to anti-neoplastic treatment, or alternatively, recurs or relapses after having responded favorably to anti-neoplastic treatment. Thus, as used herein, a "treatment-refractory cancer" refers to a cancer that does not respond favorably to treatment, or is resistant to treatment, or alternatively, recurs or relapses after having responded favorably to treatment. For example, such prior treatment may be chemotherapy, or may be an immunotherapy regimen comprising administration of a monoclonal antibody that specifically binds to PD-1, PD-L1 or CTLA4.

The use of the singular term "comprising" when used in the claims and/or in this specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

It is contemplated that any embodiment discussed herein can be practiced with respect to any method or composition of the present invention, and vice versa. Furthermore, the compositions and kits of the present invention can be used to achieve the methods of the present invention.

Throughout this specification, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being used to determine the value.

Although the present disclosure supports definitions referring to only one alternative and "and/or," the use of the term "or" in the claims is intended to mean "and/or" unless it is specifically indicated to refer to an alternative alone or that the alternatives are mutually exclusive.

As used in this specification and claims(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or expansive and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

It has been found that combination therapy with anticancer vaccinia virus and a checkpoint inhibitor results in unexpected improvements in the treatment of cancer. When the agents are administered in combination and the anticancer vaccinia virus is administered, the agents cooperate and even synergistically interact to provide significantly improved antitumor effects compared to single administration of either agent. Surprisingly, these effects are not significantly observed when the agents are administered sequentially. In some embodiments, when the replicating anticancer virus is administered intratumorally, the combination therapy provides a synergistic effect. In some embodiments, when the replicating anticancer virus is administered intravenously, the combination therapy provides a synergistic effect. In some embodiments, when the replicating anticancer virus is administered intraperitoneally, the combination therapy provides a synergistic effect. In some embodiments, when the replicating anticancer virus is delivered exclusively via intratumoral administration, the combination therapy provides a synergistic effect. In some embodiments, when the replicating anticancer virus is delivered exclusively via intraarterial administration, the combination therapy provides a synergistic effect. In some embodiments, the checkpoint inhibitor is administered systemically. In some embodiments, when the replicating oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically, the combination therapy provides a synergistic effect. In some embodiments, when the replicating oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically, the combination therapy provides a synergistic effect. In some embodiments, when the replicating oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically, the combination therapy provides a synergistic effect. In some embodiments, when the replicating oncolytic virus is administered intraarterially and the checkpoint inhibitor is administered systemically, the combination therapy provides a synergistic effect.

Additionally, we found that oncolytic vaccinia virus sensitizes tumors to treatment with checkpoint inhibitors by upregulating the expression of checkpoint proteins such as PD-1, PD-L1, CTLA-4, TIM3, LAG3 and TIGIT in human tumors, supporting combination therapy in patients with tumors expressing the checkpoint inhibitors of the combination as well as in patients who do not express the checkpoint inhibitors of the combination or express relatively low levels of the checkpoint inhibitors.

In some embodiments, a combination therapy is provided for use in the treatment and/or prevention of cancer and/or establishment of metastasis in a mammal (preferably a human), comprising administering to the mammal (i) a replication competent oncolytic vaccinia virus and (ii) one or more immune checkpoint inhibitors in combination. In some embodiments, the replication competent oncolytic vaccinia virus is administered intratumorally. In some embodiments, the replication competent oncolytic vaccinia virus is administered intravenously. In some embodiments, the replication competent oncolytic vaccinia virus is administered intratumorally only. In some embodiments, the replication competent oncolytic vaccinia virus is administered intraarterially only. In some embodiments, when the replication competent oncolytic virus is administered via intraperitoneal administration, the combination therapy provides a synergistic effect. In some embodiments, the checkpoint inhibitor is administered systemically. In some embodiments, the replication competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replication competent oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraarterially and the checkpoint inhibitor is administered systemically.

II. anticancer vaccinia virus

Vaccinia virus is a large, complex enveloped virus with a linear double-stranded DNA genome of approximately 190 Kbp and encoding approximately 250 genes. Vaccinia is best known for its role as the vaccine that eradicated smallpox. After the eradication of smallpox, scientists explored the use of vaccinia as a tool for transferring genes into biological tissues (gene therapy and genetic engineering). Vaccinia virus is unique among DNA viruses in that it replicates exclusively in the cytoplasm of the host cell. Therefore, the large genome is required to encode the various enzymes and proteins required for viral DNA replication. During replication, vaccinia produces several different infectious forms in their envelope: intracellular mature virions (EVIV), intracellular enveloped virions (TEV), cell-associated enveloped virions (CEV), and extracellular enveloped virions (EEV). EVIV is the most common infectious form and is thought to be responsible for spreading between hosts. In contrast, CEV is thought to play a role in cell-to-cell spread, while EEV is thought to be important for long-range transmission within the host organism.

Any known anti-cancer strain of vaccinia virus can be administered in combination with a checkpoint inhibitor according to the methods described herein. In a preferred embodiment, the replicative anti-cancer vaccinia virus is a Copenhagen, Western Reserve, Lister or Wyeth strain, most preferably a Western Reserve or Wyeth strain. The genome of the Western Reserve vaccinia strain has been sequenced (Accession No. AY243312). In some embodiments, the replicative anti-cancer vaccinia virus is a Copenhagen strain. In some embodiments, the replicative anti-cancer vaccinia virus is a Western Reserve strain. In some embodiments, the replicative anti-cancer vaccinia virus is a Lister strain. In some embodiments, the replicative anti-cancer vaccinia virus is a Wyeth strain.

The replicative oncolytic vaccinia virus can be engineered to lack one or more functional genes to increase the oncolytic selectivity of the virus. In some preferred embodiments, the oncolytic vaccinia virus is engineered to lack thymidine kinase (TK) activity. TK-deficient vaccinia viruses require thymidine triphosphate for DNA synthesis, which results in favorable replication in dividing cells (particularly cancer cells). In another aspect, the oncolytic vaccinia virus can be engineered to lack vaccinia virus growth factor (VGF). This secreted protein is produced early in the infection process and acts as a mitogen to prime surrounding cells for infection. In another aspect, the oncolytic vaccinia virus can be engineered to lack both VFG and TK activity. In another aspect, the oncolytic vaccinia virus can be engineered to lack one or more genes involved in evading a host interferon (IFN) response, such as E3L, K3L, B18R, or B8R. In some preferred embodiments, the replicative oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister, or Wyeth strain and lacks a functional TK gene. In other embodiments, the oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister, or Wyeth strain and lacks a functional B18R and/or B8R gene. In some embodiments, the replicative oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister, or Wyeth strain and lacks a functional TK gene. In some embodiments, the replicative oncolytic vaccinia virus is a Western Reserve strain and lacks a functional TK gene. In some embodiments, the replicative oncolytic vaccinia virus is a Copenhagen strain and lacks a functional TK gene. In some embodiments, the replicative oncolytic vaccinia virus is a Lister strain and lacks a functional TK gene. In some embodiments, the replicative oncolytic vaccinia virus is a Wyeth strain and lacks a functional TK gene. In some embodiments, the oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister, or Wyeth strain that lacks a functional B18R and/or B8R gene. In some embodiments, the oncolytic vaccinia virus is a Western Reserve strain that lacks a functional B18R and/or B8R gene. In some embodiments, the oncolytic vaccinia virus is a Copenhagen strain that lacks a functional B18R and/or B8R gene. In some embodiments, the oncolytic vaccinia virus is a Lister strain that lacks a functional B18R and/or B8R gene. In some embodiments, the oncolytic vaccinia virus is a Wyeth strain that lacks a functional B18R and/or B8R gene. In some embodiments, the replicating oncolytic vaccinia virus is a Western Reserve, Copenhagen, Lister, or Wyeth strain that lacks a functional TK gene as well as a functional B18R and/or B8R gene. In some embodiments, the replicative oncolytic vaccinia virus comprises a functional 14L and/or F4L gene. In some embodiments, the replicative oncolytic vaccinia virus does not express a chemokine (e.g., the vaccinia virus does not express CXCL-11).

In some embodiments, the replicative oncolytic vaccinia virus of the combination comprises functional 14L and/or F4L genes.

In another embodiment, the replicative oncolytic vaccinia virus of the combination does not express a chemokine (e.g., the vaccinia virus does not express CXCL-11).

The heterologous sequence (e.g., encoding a cytokine and/or tumor antigen) can be placed under the control of a vaccinia virus promoter and integrated into the genome of the vaccinia virus. Alternatively, expression of the heterologous sequence can be achieved by transfecting cells infected with vaccinia virus with a shuttle vector or plasmid containing a vaccinia promoter-control sequence and introducing the heterologous sequence by homologous recombination, such as those found in Table 1 of Current Techniques in Molecular Biology, (Ed. Ausubel, et al.) Unit 16.17.4 (1998). The strong late vaccinia virus promoter is preferred when high-level expression is desired. Early and intermediate promoters can also be used. In some embodiments, the heterologous sequence is under the control of a vaccinia virus promoter containing early and late promoter elements. Suitable early promoters include, but are not limited to, promoters of vaccinia virus genes encoding a 42K, 19K or 25K polypeptide. Suitable early late promoters include, but are not limited to, promoters of vaccinia virus genes encoding a 7.5K polypeptide. Suitable late promoters include, but are not limited to, promoters of vaccinia virus genes encoding an 11K or 28K polypeptide. In a related embodiment, the heterologous sequence is inserted into the TK and/or VGF sequences to inactivate the TK and/or VGF sequences.

In some embodiments, the replicating oncolytic vaccinia virus described herein is administered in combination with one or more checkpoint inhibitors. In some embodiments, the replicating oncolytic vaccinia virus described herein is administered intratumorally in combination with one or more checkpoint inhibitors. In some embodiments, the replicating oncolytic vaccinia virus described herein is administered intravenously (IV; or intravascularly) in combination with one or more checkpoint inhibitors. In some embodiments, the replicating oncolytic vaccinia virus described herein is administered intraperitoneally (IP) in combination with one or more checkpoint inhibitors. In some embodiments, the replicating oncolytic vaccinia virus described herein is administered intraarterially in combination with one or more checkpoint inhibitors. In some embodiments, the replicating oncolytic virus is delivered exclusively via intratumoral administration. In some embodiments, the replicating oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraarterially and the checkpoint inhibitor is administered systemically. Intratumoral administration generally involves injection into the tumor mass or into the tumor-associated vasculature. In certain aspects, the tumor is imaged prior to or during administration of the virus. Intravascular administration generally involves injection into the vasculature and is a form of systemic administration. Intraperitoneal administration generally involves injection into the peritoneum (e.g., a body cavity). In some embodiments, the replicating oncolytic vaccinia virus described herein is administered in combination with one or more checkpoint inhibitors, both of which are administered systemically, e.g., by IV administration. In some embodiments, the replicating oncolytic vaccinia virus described herein is administered in combination with one or more checkpoint inhibitors, wherein the replicating oncolytic vaccinia virus is administered intratumorally and the one or more checkpoint inhibitors are administered systemically, for example, by IV administration. In some embodiments, the replicating oncolytic vaccinia virus described herein is administered in combination with one or more checkpoint inhibitors, wherein the replicating oncolytic vaccinia virus is administered intraperitoneally and the one or more checkpoint inhibitors are administered systemically, for example, by IV administration. In some embodiments, the replicating oncolytic vaccinia virus described herein is administered in combination with one or more checkpoint inhibitors, wherein the replicating oncolytic vaccinia virus is administered intraarterially and the one or more checkpoint inhibitors are administered systemically, for example, by IV administration.

The anticancer vaccinia virus as described herein may be administered as a single dose or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8 or more times). The virus is present in an amount of 1×10 ⁵ plaque forming units (PFU), 5×10 ⁵ PFU, 1×10 ⁶ PFU, at least 1×10 ⁶ PFU, 5×10 ⁶ or about 5×10 ⁶ PFU, 1×10 ⁷ , at least 1×10 ⁷ PFU, 1×10 ⁸ or about 1×10 ⁸ PFU, at least 1×10 ⁸ PFU, about or at least 5×10 ⁸ PFU, 1×10 ⁹ or at least 1×10 ⁹ PFU, 5×10 ⁹ or at least 5×10 ⁹ PFU, 1×10 ¹⁰ PFU or at least 1×10 ¹⁰ PFU, 5×10 ¹⁰ or at least 5×10 ¹⁰ PFU, 1×10 ¹¹ or at least 1×10 ¹¹ , 1×10 ¹² or at least 1×10 ¹² , 1×10 ¹³ or at least 1×10 ¹³ may be administered. For example, the virus can be administered at a dosage of about 10 ⁶ to 10 ¹³ pfu, about 10 ⁷ to 10 ¹³ pfu, about 10 ⁸ to 10 ¹³ pfu, about 10 ⁹ to 10 ¹² pfu, about 10 ⁸ to 10 ¹² pfu, about 10 ⁷ to 10 ¹² pfu, about 10 ⁶ to 10 ¹² pfu, about 10 ⁶ to 10 ⁹ pfu, about 10 ⁶ to 10 ⁸ pfu, about 10 ⁷ to 10 ¹⁰ pfu, about 10 ⁷ to 10 ⁹ pfu, about 10 ⁸ to 10 ¹⁰ pfu, or about 10 ⁸ to 10 ⁹ pfu, preferably, the virus is administered at a dosage of at least 10 ⁷ pfu, 10 ⁷ to 10 Administered in dosages of ¹⁰ pfu, 10 ⁷ to 10 ⁹ pfu, 10 ⁷ to 10 ⁸ pfu, 10 ⁸ to 10 ¹⁰ pfu, 10 ⁸ to 10 ⁹ pfu or 10 ⁹ to 10 ¹⁰ pfu.

A single dose of virus is intended to refer to an amount administered to a subject or tumor over a period of 0.1, 0.5, 1, 2, 5, 10, 15, 20 or 24 hours (including all values in between). The dose may be spread over time or by separate injections. Typically, multiple doses are administered to the same general target area, such as proximal to the tumor. In certain aspects, the virus dose is delivered by an injection device comprising a syringe or a single needle or multiple prongs coupled to a syringe, a single port needle or multiple ports or a combination thereof. A single dose of vaccinia virus may be administered or multiple doses may be administered over a treatment period which may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more. For example, vaccinia virus can be administered every other day, every week, every other week, or every three weeks for periods of 1, 2, 3, 4, 5, or 6 months or longer.

Vaccinia virus can be propagated using the method described by Earl and Moss in Ausubel et al, 1994 or the method described in WIPO Publication No. WO2013/022764, both of which are incorporated herein by reference.

III. Immune checkpoint inhibitors (ICIs)

Immune checkpoint proteins interact with specific ligands that signal T cells to inhibit T cell function. Cancer cells take advantage of this by inducing high levels of checkpoint proteins on their surface, thereby suppressing antitumor immune responses.

An immune checkpoint inhibitor (also referred to as an ICI) for use in the pharmaceutical combinations described herein is any compound capable of inhibiting the function of an immune checkpoint protein. Inhibition includes not only a reduction in function but also a complete blockade. In particular, the immune checkpoint protein is a human checkpoint protein. Therefore, the immune checkpoint inhibitor is preferably an inhibitor of a human immune checkpoint.

Checkpoint proteins include, but are not limited to, CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, BTLA, TIGIT, and/or IDO. It is recognized in the art that pathways involving LAG3, BTLA, B7-H3, B7-H4, TIM3, and KIR constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see, e.g., Pardoll, 2012, Nature Rev Cancer 12:252-264; Mellman et al., 2011, Nature 480:480-489). In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, BTLA, TIGIT, and/or IDO. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, and/or TIM3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIGIT. In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM3.

In some embodiments, the immune checkpoint inhibitor of the combination is an antibody. The term "antibody" as used herein includes, for example, naturally occurring and engineered antibodies capable of binding to a target immune checkpoint or epitope (e.g., having an antigen-binding portion), as well as full-length antibodies or functional fragments or analogs thereof. Antibodies for use in accordance with the methods described herein can be of any origin, including but not limited to, human, humanized, or chimeric, can have any isotype with a preference for the IgG1 or IgG4 isotype, and can further be glycosylated or non-glycosylated. The term antibody also includes bispecific or multispecific antibodies, so long as the antibody(ies) exhibit the binding specificities described herein.

A humanized antibody refers to a non-human (e.g., murine, rat, etc.) antibody whose protein sequence has been altered to increase its similarity to human antibodies. A chimeric antibody refers to an antibody comprising a non-human antibody that comprises one or more elements(s) of one species and one or more elements(s) of another species, such as at least a portion of the constant region (Fc) of a human immunoglobulin.

A number of forms of antibodies can be engineered for use in the combinations of the present invention, notably a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CH1 domains), a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by at least one disulfide bridge at the hinge region), a Fd fragment (composed of the VH and CH1 domains), a Fv fragment (composed of the VL and VH domains of a single arm of an antibody), a dAb fragment (composed of a single variable domain fragment (either the VH or VL domain)), a Fv fragment that is ultimately fused together with a linker to form a single protein chain, and a single-chain Fv (scFv) comprising two domains, VL and VH.

In some embodiments, the immune checkpoint protein inhibitor (also referred to as an ICI) of the combination therapy is an antibody or fragment thereof that specifically binds to an immune checkpoint protein selected from the group consisting of CTLA4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, TIM3, GAL9, LAG3, VISTA, KIR, BTLA and TIGIT. In a particularly preferred embodiment, the immune checkpoint inhibitor is a monoclonal antibody, a fully human antibody, a chimeric antibody, a humanized antibody or a fragment thereof that can at least partially antagonize CTLA4, PD-1, PD-L1, PD-L2, TIM3, LAG3 or TIGIT. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds CTLA4. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds PD-1. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds PD-L1. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds PD-L2. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds B7-H3. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds B7-H4. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds TIM3. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds GAL9. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds LAG3. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds VISTA. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds KIR. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds BTLA. In some embodiments, the immune checkpoint protein inhibitor of the combination therapy is an antibody or fragment thereof that specifically binds TIGIT.

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a CTLA-4 inhibitor, preferably a monoclonal antibody that specifically binds to (inhibits) CTLA-4. The complete human CTLA-4 nucleic acid sequence can be found under GenBank Accession Number LI 5006. Monoclonal antibodies that specifically bind to CTLA4 include ipilimumab (Yervoy®; BMS) and tremelimumab (AstraZeneca/MedImmune), as well as antibodies disclosed in U.S. Patent Application Publication Nos. 2005/0201994, 2002/0039581, and 2002/086014 (each of which is incorporated herein by reference), U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720, 6,682,736, 6,207,156, 5,977,318, 6,682,736, Including, but not limited to, antibodies disclosed in U.S. Pat. Nos. 7,109,003, 7,132,281, and 8,491,895 (each of which is incorporated herein by reference), or antibodies comprising the heavy and light chain variable regions of any of these antibodies.

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a PD-1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-1. The complete nucleotide and amino acid sequence of human PD-1 can be found under GenBank Accession Numbers U64863 and NP_005009.2. Monoclonal antibodies to PD-1 include lambrolizumab (e.g., disclosed as hPD109A and its humanized derivatives h409A11, h409A16 and h409A17 in U.S. Pat. No. 8,354,509, which is incorporated herein by reference), nivolumab (Opdivo®; Bristol-Myers Squibb; codename BMS-936558) disclosed in U.S. Pat. No. 8,008,449, which is incorporated herein by reference), pembrolizumab (Keytruda®) and pidilizumab (CT-011; disclosed in Rosenblatt et al. , Immunother. 34:409-418 (2011)) or antibodies comprising the heavy and light chain regions of these antibodies. Including, but not limited to, other anti-PD-1 antibodies are described, for example, in WO2004/004771, WO2004/056875, WO2006/121168, WO2008/156712, WO2009/014708, WO2009/114335, WO2013/043569 and WO2014/047350. In a related embodiment, the checkpoint inhibitor of the pharmaceutical combination is an anti-PD-1 fusion protein, such as AMP-224 (composed of the extracellular domain of PD-L2 and the Fc region of human IgG1).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a PD-L1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-L1. Monoclonal antibodies to PD-L1 include pembrolizumab (MK-3475, disclosed in WO2009/114335), BMS-936559 (MDX-1105), atezolizumab (Genentech/Roche; MPDL33280A) disclosed in U.S. Pat. No. 8,217,149, the contents of which are incorporated herein by reference, durvalumab (AstraZeneca/Medimune; MEDI4736) disclosed in U.S. Pat. No. 8,779,108, the contents of which are incorporated herein by reference, MIH1 (Affymetrix (16.5983.82) available through eBioscience) and avelumab (MSB0010718C; Merck KGaA) or any Include, but are not limited to, antibodies comprising the heavy and light chain variable regions of these antibodies. In a related embodiment, the immune checkpoint inhibitor is an anti-PD-L1 fusion protein, such as a PD-L2-Fc fusion protein known as AMP-224 (disclosed in the literature [Mkritchyan M., et al., J. Immunol., 189:2338-47 (2010)]).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister, or Copenhagen vaccinia virus strain and a PD-L2 inhibitor, such as MIH18 (described in Pfistershammer et al. , Eur J Immunol. 36: 1104-1113 (2006)).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a LAG3 inhibitor, such as soluble LAG3 (IMP321 or a LAG3-Ig as disclosed in U.S. Patent Application Publication No. 2011-0008331, incorporated herein by reference, and Brignon et al. , Clin. Cancer Res. 15:6225-6231 (2009)), IMP701 or other humanized antibodies that block human LAG3 as disclosed in U.S. Patent Application Publication No. 2010-0233183, incorporated herein by reference, U.S. Pat. No. 5,773,578, incorporated herein by reference, or BMS-986016 or other fully human antibodies that block LAG3 as disclosed in U.S. Patent Application Publication No. 2011-0150892, incorporated herein by reference.

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a BLTA inhibitor, such as antibody 4C7 disclosed in U.S. Pat. No. 8,563,694, which is incorporated herein by reference.

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a B7H4 checkpoint inhibitor, e.g., an antibody disclosed in U.S. Patent Application Publication No. 2014/0294861, which is incorporated herein by reference, or a soluble recombinant form of B7H4, e.g., those disclosed in U.S. Patent Application Publication No. 20120177645, which is incorporated herein by reference.

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a B7-H3 checkpoint inhibitor, such as BRCA84D or a derivative thereof as disclosed in U.S. Patent Application Publication No. 20120294796, which is incorporated herein by reference, and the antibody MGA271.

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a TIM3 checkpoint inhibitor, such as an antibody disclosed in U.S. Pat. No. 8,841,418, which is incorporated herein by reference, or the anti-human TIM3 blocking antibody F38-2E2 disclosed by Jones et al., J. Exp. Med., 205(12):2763-79 (2008).

In some embodiments, the pharmaceutical combination comprises Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a KIR checkpoint inhibitor, such as the antibody lililumab (described in Romagne et al. , Blood, 114(13):2667-2677 (2009)).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a TIGIT inhibitor. The TIGIT checkpoint inhibitor preferably inhibits the interaction of TIGIT with the poliovirus receptor (CD155) and includes, but is not limited to, antibodies targeting human TIGIT, such as those disclosed in U.S. Patent No. 9,499,596 (incorporated herein by reference) and U.S. Patent Application Publication Nos. 20160355589, 20160176963 (incorporated herein by reference) and poliovirus receptor variants, such as those disclosed in U.S. Patent No. 9,327,014 (incorporated herein by reference).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and an IDO inhibitor. IDO is recognized as an immune checkpoint protein whose expression on tumor cells contributes to immune tolerance by arresting effector T cells. IDO is thought to contribute to resistance to anti-CLTA-4 therapy. Inhibitors of IDO for use in accordance with the methods described herein include, but are not limited to, tryptophan mimetics, such as D-1MT (the D isoform (MT) of 1-methyl-DL-tryptophan), L-1MT (the L isoform of MT), MTH-Trp (methylthiohydantoin-dl-tryptophan; a transcriptional inhibitor of IDO), and β-carbolines, indole mimetics, such as naphthoquinone based agents, S-allyl-brassinine, S-benzyl-brassinine, 5-bromo-brassinine, as well as phenylimidazole based agents, 4-phenylimidazole, exiguamine A, epacadostat, rosmarinic acid, norhamane and NSC401366. Preferred IDO inhibitors include INCB 024360 (epacadostat; 1,2,5-oxadiazole-3-carboximidamide, 4-((2-((aminosulfonyl)amino)ethyl)amino)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-, (C(Z))-; Incyte), indoximod (NLG2101; D-1MT; NewLink Genetics), IDO peptide vaccine (Copenhagen University) and NLG919 (NewLink Genetics).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain, a PD-1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-1 and a CTLA-4 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) CTLA-4. The complete nucleotide and amino acid sequence of human PD-1 can be found under GenBank Accession Numbers U64863 and NP_005009.2. Monoclonal antibodies to PD-1 include, but are not limited to, lambrolizumab (disclosed, e.g., as hPD109A and its humanized derivatives h409A11, h409A16 and h409A17, in U.S. Pat. No. 8,354,509, which is incorporated herein by reference), nivolumab (Opdivo®; Bristol-Myers Squibb; codename BMS-936558, which is disclosed in U.S. Pat. No. 8,008,449, which is incorporated herein by reference), pembrolizumab (Keytruda®), and pidilizumab (CT-011; disclosed in Rosenblatt et al., Immunother. 34:409-418 (2011)) or antibodies comprising the heavy and light chain regions of these antibodies. Other anti-PD-1 antibodies are described, for example, in WO2004/004771, WO2004/056875, WO2006/121168, WO2008/156712, WO2009/014708, WO2009/114335, WO2013/043569 and WO2014/047350. In a related embodiment, the checkpoint inhibitor of the pharmaceutical combination is an anti-PD-1 fusion protein, such as AMP-224 (composed of the extracellular domain of PD-L2 and the Fc region of human IgG1). The fully human CTLA-4 nucleic acid sequence can be found in GenBank under Accession No. LI 5006. Monoclonal antibodies that specifically bind to CTLA4 include ipilimumab (Yervoy®; BMS) and tremelimumab (AstraZeneca/Medimune), as well as antibodies disclosed in U.S. Patent Application Publication Nos. 2005/0201994, 2002/0039581, and 2002/086014, the contents of which are incorporated herein by reference, and U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720, 6,682,736, 6,207,156, 5,977,318, 6,682,736, 7,109,003, 7,132,281, and Includes, but is not limited to, antibodies disclosed in U.S. Pat. No. 8,491,895 (the contents of each of which are incorporated herein by reference) or antibodies comprising the heavy and light chain variable regions of any of these antibodies.

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain, a PD-L1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-L1, and a CTLA-4 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) CTLA-4. Monoclonal antibodies to PD-L1 include, but are not limited to, pembrolizumab (MK-3475, disclosed in WO2009/114335), BMS-936559 (MDX-1105), atezolizumab (Genentech/Roche; MPDL33280A) disclosed in U.S. Pat. No. 8,217,149 (the contents of which are herein incorporated by reference), durvalumab (AstraZeneca/Medimune; MEDI4736) disclosed in U.S. Pat. No. 8,779,108, which is incorporated by reference herein), MIH1 (Affymetrix (16.5983.82) available through eBiosciences) and avelumab (MSB0010718C; Merck KGaA) or antibodies comprising the heavy and light chain variable regions of any of these antibodies. In a related embodiment, the immune checkpoint inhibitor is an anti-PD-L1 fusion protein, such as a PD-L2-Fc fusion protein known as AMP-224 (disclosed in the literature [Mkritchyan M., et al., J. Immunol., 189:2338-47 (2010)]). The fully human CTLA-4 nucleic acid sequence can be found under GenBank Accession No. LI 5006. Monoclonal antibodies that specifically bind to CTLA4 include ipilimumab (Yervoy®; BMS) and tremelimumab (AstraZeneca/Medimune), as well as antibodies disclosed in U.S. Patent Application Publication Nos. 2005/0201994, 2002/0039581, and 2002/086014 (the contents of each of which are incorporated herein by reference), and U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720, 6,682,736, 6,207,156, 5,977,318, 6,682,736, 7,109,003, 7,132,281, and Includes, but is not limited to, antibodies disclosed in U.S. Pat. No. 8,491,895 (the contents of each of which are incorporated herein by reference), or antibodies comprising the heavy and light chain variable regions of any of these antibodies.

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain, a PD-1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-1, and a LAG3 inhibitor, such as soluble LAG3 (IMP321 or LAG3-Ig disclosed in U.S. Patent Application Publication No. 2011-0008331, incorporated herein by reference, and Brignon et al., Clin. Cancer Res. 15:6225-6231 (2009)), IMP701 or other humanized antibodies that block human LAG3 disclosed in U.S. Patent Application Publication No. 2010-0233183, incorporated herein by reference, U.S. Pat. No. 5,773,578, incorporated herein by reference, or BMS-986016 or U.S. Patent Application Publication No. 2010-0233183, incorporated herein by reference, or other humanized antibodies that block human LAG3 disclosed in U.S. Patent No. 5,773,578, incorporated herein by reference. Other fully human antibodies that block LAG3 are described in Korean Patent No. 2011-0150892. The complete nucleotide and amino acid sequence of human PD-1 can be found under GenBank Accession No. U64863 and NP_005009.2. Monoclonal antibodies to PD-1 include, but are not limited to, lambrolizumab (disclosed, e.g., as hPD109A and its humanized derivatives h409A11, h409A16 and h409A17, in U.S. Pat. No. 8,354,509, which is incorporated herein by reference), nivolumab (Opdivo®; Bristol-Myers Squibb; codename BMS-936558, which is disclosed in U.S. Pat. No. 8,008,449, which is incorporated herein by reference), pembrolizumab (Keytruda®), and pidilizumab (CT-011; disclosed in Rosenblatt et al., Immunother. 34:409-418 (2011)) or antibodies comprising the heavy and light chain regions of these antibodies. Other anti-PD-1 antibodies are described, for example, in WO2004/004771, WO2004/056875, WO2006/121168, WO2008/156712, WO2009/014708, WO2009/114335, WO2013/043569 and WO2014/047350. In a related embodiment, the checkpoint inhibitor of the pharmaceutical combination is an anti-PD-1 fusion protein, such as AMP-224 (composed of the extracellular domain of PD-L2 and the Fc region of human IgG1).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain, a PD-L1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-L1, and a LAG3 inhibitor, such as soluble LAG3 (IMP321 or LAG3-Ig disclosed in U.S. Patent Application Publication No. 2011-0008331, incorporated herein by reference, and Brignon et al., Clin. Cancer Res. 15:6225-6231 (2009)), IMP701 or other humanized antibodies that block human LAG3 described in U.S. Patent Application Publication No. 2010-0233183, incorporated herein by reference, U.S. Pat. No. 5,773,578, incorporated herein by reference, or BMS-986016 or U.S. Patent Application Publication No. 2010-0233183, incorporated herein by reference, or other humanized antibodies that block human LAG3 described in U.S. Patent No. 5,773,578, incorporated herein by reference. Other fully human antibodies that block LAG3 are described in No. 2011-0150892. Monoclonal antibodies to PD-L1 include, but are not limited to, pembrolizumab (MK-3475, disclosed in WO2009/114335), BMS-936559 (MDX-1105), atezolizumab (Genentech/Roche; MPDL33280A) disclosed in U.S. Pat. No. 8,217,149 (the contents of which are herein incorporated by reference), durvalumab (AstraZeneca/Medimune; MEDI4736) disclosed in U.S. Pat. No. 8,779,108, which is incorporated by reference herein), MIH1 (Affymetrix (16.5983.82) available through eBiosciences) and avelumab (MSB0010718C; Merck KGaA) or antibodies comprising the heavy and light chain variable regions of any of these antibodies. In a related embodiment, the immune checkpoint inhibitor is an anti-PD-L1 fusion protein, such as a PD-L2-Fc fusion protein known as AMP-224 (disclosed in the literature [Mkritchyan M., et al., J. Immunol., 189:2338-47 (2010)]).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain, a PD-1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-1, and a TIM3 checkpoint inhibitor, such as an antibody disclosed in U.S. Pat. No. 8,841,418, which is incorporated herein by reference, or the anti-human TIM3 blocking antibody F38-2E2 disclosed by Jones et al., J. Exp. Med., 205(12):2763-79 (2008). The complete nucleotide and amino acid sequence of human PD-1 can be found under GenBank Accession Nos. U64863 and NP 005009.2. Monoclonal antibodies to PD-1 include, but are not limited to, lambrolizumab (disclosed, e.g., as hPD109A and its humanized derivatives h409A11, h409A16 and h409A17, in U.S. Pat. No. 8,354,509, which is incorporated herein by reference), nivolumab (Opdivo®; Bristol-Myers Squibb; codename BMS-936558, which is disclosed in U.S. Pat. No. 8,008,449, which is incorporated herein by reference), pembrolizumab (Keytruda®), and pidilizumab (CT-011; disclosed in Rosenblatt et al., Immunother. 34:409-418 (2011)) or antibodies comprising the heavy and light chain regions of these antibodies. Other anti-PD-1 antibodies are described, for example, in WO2004/004771, WO2004/056875, WO2006/121168, WO2008/156712, WO2009/014708, WO2009/114335, WO2013/043569 and WO2014/047350. In a related embodiment, the checkpoint inhibitor of the pharmaceutical combination is an anti-PD-1 fusion protein, such as AMP-224 (composed of the extracellular domain of PD-L2 and the Fc region of human IgG1).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain, a PD-L1 inhibitor, preferably a monoclonal antibody that specifically binds to (and inhibits) PD-L1, and a TIM3 checkpoint inhibitor, such as an antibody disclosed in U.S. Pat. No. 8,841,418, which is incorporated herein by reference, or the anti-human TIM3 blocking antibody F38-2E2 disclosed by Jones et al., J. Exp. Med., 205(12):2763-79 (2008). Monoclonal antibodies to PD-L1 include, but are not limited to, pembrolizumab (MK-3475, disclosed in WO2009/114335), BMS-936559 (MDX-1105), atezolizumab (Genentech/Roche; MPDL33280A) disclosed in U.S. Pat. No. 8,217,149 (the contents of which are herein incorporated by reference), durvalumab (AstraZeneca/Medimune; MEDI4736) disclosed in U.S. Pat. No. 8,779,108, which is incorporated by reference herein), MIH1 (Affymetrix (16.5983.82) available through eBiosciences) and avelumab (MSB0010718C; Merck KGaA) or antibodies comprising the heavy and light chain variable regions of any of these antibodies. In a related embodiment, the immune checkpoint inhibitor is an anti-PD-L1 fusion protein, such as a PD-L2-Fc fusion protein known as AMP-224 (disclosed in the literature [Mkritchyan M., et al., J. Immunol., 189:2338-47 (2010)]).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and a TIGIT inhibitor. The TIGIT checkpoint inhibitor preferably inhibits the interaction of TIGIT with the poliovirus receptor (CD155) and includes, but is not limited to, antibodies targeting human TIGIT, such as those disclosed in U.S. Patent No. 9,499,596 (incorporated herein by reference) and U.S. Patent Application Publication Nos. 20160355589, 20160176963 (incorporated herein by reference) and poliovirus receptor variants, such as those disclosed in U.S. Patent No. 9,327,014 (incorporated herein by reference).

In some embodiments, the pharmaceutical combination comprises a Western Reserve, Wyeth, Lister or Copenhagen vaccinia virus strain and an IDO inhibitor (indoleamine-pyrrole 2,3-dioxygenase. The IDO inhibitor comprises a metabolic inhibitor, preferably one which inhibits a metabolic pathway, see Norharmane (Chiarugi A, et al , "Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-gamma-activated macrophages", Journal of Leukocyte Biology . 68 (2): 260-6. (2000)), rosmarinic acid (Lee HJ, et al , "Rosmarinic acid inhibits indoleamine 2,3-dioxygenase expression in murine dendritic cells", Biochemical Pharmacology. 73 (9): 1412-21 (2007)] 참조), COX-2 저해제(문헌[Cesario A, et al., "The interplay between indoleamine 2,3-dioxygenase 1(IDO1) and cyclooxygenase (COX)-2 in chronic inflammation and cancer", Current Medicinal Chemistry. 18 (15): 2263-71 (2011)]), 1-메틸트립토판(문헌[Hou DY, et al., "Inhibition of indoleamine 2,3-dioxygenase in dendritic cells by stereoisomers of 1-methyl-tryptophan correlates with antitumor responses". Cancer Research. 67 (2): 792-801 (2007) 및 Chauhan N, et al., (April 2009). "Reassessment of the reaction mechanism in the heme di oxygenases". Journal of the American Chemical Society. 131 (12): 4186-7 (2009)]) (including, for example, a specific racemic 1-methyl-D-tryptophan (also known as indoximod) (a clinical trial candidate), epacadostat (INCB24360), noboximod (GDC-0919) (see Jochems C, et al., "The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells", Oncotarget. 7 (25): 37762-37772. (2016)]), and or BMS-986205. In some embodiments, the IDO inhibitor is selected from the group consisting of norhamane, rosmarinic acid, a COX-2 inhibitor, 1-methyltryptophan, indoximod, epacadostat (INCB24360), noboximod (GDC-0919), and/or BMS-986205.

As will be known to those skilled in the art, alternative and/or equivalent names may be used for any of the above-mentioned antibodies. Such alternative and/or equivalent names are interchangeable within the context of the present invention.

In some aspects, the pharmaceutical combinations described herein comprise (i) more than one immune checkpoint inhibitor and (ii) a replicating oncolytic vaccinia virus. In a preferred embodiment, the PD-1 inhibitor and the CTLA-4 inhibitor are administered in combination with the vaccinia virus. Other examples include, but are not limited to, the concomitant administration of a LAG3 inhibitor and a PD-1 inhibitor and the vaccinia virus, or the concomitant administration of a LAG3 inhibitor and a PD-L1 inhibitor. Other examples include the concomitant administration of an IDO inhibitor and a CTLA-4 inhibitor and/or a PD-1 inhibitor. In some embodiments, the IDO inhibitor is selected from the group consisting of norhamane, rosmarinic acid, a COX-2 inhibitor, 1-methyltryptophan, indoximod, epacadostat (INCB24360), naboximod (GDC-0919), and/or BMS-986205.

IV. Cytokine

In some embodiments, the replicative anti-cancer vaccinia virus of the pharmaceutical combination comprises a heterologous sequence encoding a cytokine, wherein the cytokine is expressed by the virus.

In some embodiments, a replicating oncolytic vaccinia virus is provided that is engineered to express a cytokine selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin (IL-5), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-24 (IL-24), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). In particularly preferred embodiments, the replicating oncolytic vaccinia virus is a Wyeth, Western Reserve, Copenhagen or Lister strain. In some embodiments, the replicating oncolytic vaccinia virus is engineered to express GM-CSF. In some embodiments, the replicating oncolytic vaccinia virus is engineered to express interleukin-2 (IL-2). In some embodiments, the replicating oncolytic vaccinia virus is engineered to express interleukin-4 (IL-4). In some embodiments, the replicating oncolytic vaccinia virus is engineered to express interleukin-5 (IL-5). In some embodiments, the replicating oncolytic vaccinia virus is engineered to express interleukin-7 (IL-7). In some embodiments, the replicating oncolytic vaccinia virus is engineered to express interleukin-12 (IL-12). In some embodiments, the replicating oncolytic vaccinia virus is engineered to express interleukin-15 (IL-15). In some embodiments, the replicating oncolytic vaccinia virus is engineered to express interleukin-18 (IL-18). In some embodiments, the replicating oncolytic vaccinia virus is engineered to express interleukin-21 (IL-21). In some embodiments, the replicating oncolytic vaccinia virus is engineered to express interleukin-24 (IL-24), interferon-γ (IFN-γ). In some embodiments, the replicating oncolytic vaccinia virus is engineered to express tumor necrosis factor-α (TNF-α). In some embodiments, the replicating oncolytic vaccinia virus is mJX594 engineered to express GM-CSF.

V. Tumor antigen

In some embodiments, the replicating oncolytic vaccinia virus comprises a heterologous nucleic acid sequence encoding a tumor antigen and optionally a cytokine, wherein the tumor antigen and optionally the cytokine are expressed in a cell infected with the virus, preferably a tumor cell. The tumor antigens include tumor-specific antigens and tumor-associated antigens. The replication-competent oncolytic vaccinia virus can express a full-length tumor antigen or an immunogenic peptide thereof. In some embodiments, the cytokine is expressed in a cell infected with the replicating oncolytic vaccinia virus. In some embodiments, the replicating oncolytic vaccinia virus comprises a heterologous nucleic acid sequence encoding a tumor antigen and a cytokine, wherein the tumor antigen and the cytokine are expressed in a cell infected with the replicating oncolytic vaccinia virus. In some embodiments, the cell is a tumor cell.

In some embodiments, the tumor antigen is MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, N-acetylglucosamineyltransferase-V, p-15, gp100, MART-1/MelanA, TRP-1 (gp75), TRP-2, tyrosinase, cyclin-dependent kinase 4, β-catenin, MUM-1, CDK4, HER-2/neu, human papillomavirus-E6, human papillomavirus E7, CD20, carcinoembryonic antigen (CEA), epidermal growth factor receptor, MUC-1, caspase-8, CD5, mucin-1, Lewisx, CA-125, p185HER2, IL-2R, Fap-α, tenascin, metalloproteinase-associated antigen, CAMPATH-1, RCC: G-protein signaling 5. regulator of growth factor 5 (RGS5), survivin (BIRC5 = baculovirus inhibitor of apoptosis repeat-containing 5), insulin-like growth factor-binding protein 3 (IGF-BP3), thymidylate synthase (TYMS), hypoxia-inducible protein 2 = hypoxia-inducible fat droplet-associated (HIG2), matrix metallopeptidase 7 (MMP7), prunus homolog 2 (PRUNE2), RecQ protein-like (DNA helicase Q1-like) (RECQL); leptin receptor (LEPR); ERBB receptor feedback inhibitor 1 (ERRFI1); lysosomal protein transmembrane 4 alpha (LAPTM4A); RABIB, RAS oncogene family (RABIB); CD24; Homo sapiens thymosin beta 4, X-linked (TMSB4X); Homo sapiens S100 calcium binding protein A6 (S100A6); Homo sapiens adenosine A2 receptor (ADORA2B); chromosome 16 open reading frame 61 (C16orf61); ROD1 regulator of differentiation 1 (ROD1); NAD-dependent deacetylase sirtuin-2 (SIR2L); tubulin alpha 1c (TUBA1C); ATPase inhibitor factor 1 (ATPIF1); substrate antigen 2 (STAG2); and nuclear casein kinase and cyclin-dependent substrate 1 (NUCKS1). In some embodiments, the antigen is one listed in U.S. Pat. No. 9,919,047, which is incorporated herein by reference in its entirety. In some embodiments, the tumor antigen is a renal cell carcinoma tumor antigen. In some embodiments, the renal cell carcinoma tumor antigen is regulator of G-protein signaling 5 (RGS5), survivin (BIRC5 = baculovirus inhibitor of apoptosis repeat-containing 5), insulin-like growth factor-binding protein 3 (IGF-BP3), thymidylate synthase (TYMS), hypoxia-inducible protein 2, hypoxia-inducible fat droplet-associated (HIG2), matrix metallopeptidase 7 (MMP7), Prune homolog 2 (PRUNE2), RecQ protein-like (DNA helicase Q1-like) (RECQL), leptin receptor (LEPR), ERBB receptor feedback inhibitor 1 (ERRFI1), lysosomal protein transmembrane 4 alpha (LAPTM4A); Selected from the group consisting of RABIB, RAS oncogene family (RAB1B), CD24, Homo sapiens thymosin beta 4, X-linked (TMSB4X), Homo sapiens S100 calcium binding protein A6 (S100A6), Homo sapiens adenosine A2 receptor (ADORA2B), chromosome 16 open reading frame 61 (C16orf61), ROD1 regulator of differentiation 1 (ROD1), NAD-dependent deacetylase sirtuin-2 (SIR2L), tubulin alpha 1c (TUBA1C), ATPase inhibitor factor 1 (ATPIF1), substrate antigen 2 (STAG2), and nuclear casein kinase and cyclin-dependent substrate 1 (NUCKS1). In some embodiments, the tumor antigen is selected from the group consisting of KS 1/4 pan-carcinoma antigen, ovarian cancer antigen (CA125), prostatic acid phosphatase, prostate specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate specific membrane antigen, CEA, polymorphic epithelial mucin antigen, milk fat cell antigen, colorectal tumor-associated antigen (e.g., CEA, TAG-72, C017-1A, GICA 19-9, CTA-1, and LEA), Burkitt lymphoma antigen-38.13, CD19, B-lymphoma antigen-CD20, CD33, melanoma specific antigen (e.g., ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3), tumor-specific transplantation type (TSTA) of cell surface antigens (e.g., T-antigen DNA oncovirus and RNA oncovirus). Viral envelope antigens, including virus-induced tumor antigens), oncofetal antigen-alpha-fetoprotein, such as CEA of colon, oncofetal antigen of bladder tumor, differentiation antigens (e.g. human lung cancer antigen L6 and L20), antigens of fibrosarcoma, leukemia T-cell antigen-Gp37, neoglycoprotein, sphingolipids, breast cancer antigens (e.g. EGFR (epidermal growth factor receptor), HER2 antigen (p185HER2) and HER2 neu epitope), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen-APO-1, differentiation antigens (e.g. I antigen found in fetal erythrocytes, primary endoderm, I antigen found in adult erythrocytes, preimplantation embryos, I (Ma) found in gastric adenocarcinoma, M18, M39 found in breast epithelium, SSEA-1, VEP8, VEP9 found in bone marrow cells, Myl, VIM-D5, D ₁₅₆ -22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colon adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Le ^y found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, E ₁ series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 (blood group Le ^a ) found in adenocarcinoma, NS-10, CO-43 (blood group Le ^b ) found in adenocarcinoma, G49 found in EGF receptor in A431 cells, MH2 (blood group ALe ^b /Le ^y ) found in colon adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucin, in bone marrow cells T5A7 found in melanoma, R ₂₄ found in melanoma, 4.2, G _D3 , D1.1, OFA-1, G _M2 , OFA-2, G _D2 and M1:22:25:8 found in embryonal carcinoma cells, and SSEA-3 and SSEA-4 found in 4- to 8-cell stage embryos), T-cell receptor from cutaneous T-cell lymphoma, C-reactive protein (CRP), cancer antigen-50 (CA-50), cancer antigen 15-3 (CA15-3) associated with breast cancer, cancer antigen-19 (CA-19) and cancer antigen-242 associated with gastrointestinal cancer, cancer-associated antigen (CAA), chromogranin A, epithelial mucin antigen (MC5), human epithelial-specific antigen (E1A), Lewis(a) antigen, melanoma antigen, melanoma-associated antigen 100, 25 and 150, mucin-like Including, but not limited to, cancer-associated antigen, multidrug resistance-associated protein (MRPm6), multidrug resistance-associated protein (MRP41), Neu oncogene protein (C-erbB-2), neuron-specific enolase (NSE), P-glycoprotein (mdrl gene product), multidrug resistance-associated antigen, p170, multidrug resistance-associated antigen, prostate specific antigen (PSA), CD56, and NCAM.

In some embodiments, the other tumor antigens are AEVI2 (absent in melanoma 2), BMI1 (BMI1 polycomb ring finger oncogene), COX-2 (cyclooxygenase-2), EGFRvIII (epidermal growth factor receptor variant III), EZH2 (enhancer of zeste homolog 2), LICAM (human L1 cell adhesion molecule), livin, livin β, MRP-3 (multidrug resistance protein 3), nestin, OLIG2 (oligodendrocyte transcription factor), SOX2 (SRY-related HMG-box 2), ART1 (antigen recognized by T-cell 1), ART4 (antigen recognized by T-cell 4), SART1 (squamous cell carcinoma antigen recognized by T-cell 1), SART2, SART3, B-cyclin, Gli1 (glioma-associated oncogene homolog 1), Cav-1 (caveolin-1), Cathepsin B, CD74 (cluster of differentiation 74), E-cadherin (epithelial calcium-dependent adhesion), EphA2/Eck (EPH receptor A2/epithelial kinase), Fra-1/Fosl 1 (fos-related antigen 1), Ki67 (nuclear proliferation-associated antigen of antibody Ki67), Ku70/80 (human Ku heterodimer protein subunit), IL-13Ra2 (interleukin-13 receptor subunit alpha-2), NY-ESO-1 (New York esophageal squamous cell carcinoma 1), PROX1 (prospero homeobox protein 1), PSCA (prostate stem cell antigen), SOX10 (SRY-related HMG-box 10), SOX11, survivin, UPAR (urokinase-type plasminogen activator receptor), and WT-1 (Wilm's tumor protein 1).

VI. Treatment regimens and pharmaceutical formulations

The replicating anti-cancer vaccinia virus and the immune checkpoint inhibitor of the pharmaceutical combination are administered simultaneously, wherein the anti-cancer vaccinia virus is delivered by intratumoral administration. The simultaneous administration can occur, for example, by administering each agent simultaneously in the form of a single fixed combination comprising these agents or in separate formulations. In some embodiments, the replicating anti-cancer vaccinia virus of the pharmaceutical combination and the PD-1 or PD-L1 immune checkpoint inhibitor are administered simultaneously. In some embodiments, the replicating anti-cancer vaccinia virus of the pharmaceutical combination and the CTLA-4 immune checkpoint inhibitor are administered simultaneously. In some embodiments, the replicating anti-cancer vaccinia virus of the pharmaceutical combination and the TIGIT immune checkpoint inhibitor are administered simultaneously. In some embodiments, the replicating anti-cancer vaccinia virus, the PD-1 or PD-L1 immune checkpoint inhibitor, and the CTLA-4 immune checkpoint inhibitor of the pharmaceutical combination are administered simultaneously. In some embodiments, the replicating oncolytic vaccinia virus, the PD-1 or PD-L1 immune checkpoint inhibitor, and the TIGIT immune checkpoint inhibitor of the pharmaceutical combination are administered concurrently. In some embodiments, the replicating oncolytic vaccinia virus is administered by intratumoral, intravenous, intraarterial, and/or intraperitoneal administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by intratumoral administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by intravenous administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by intraperitoneal administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by intraarterial administration. In some embodiments, the replicating oncolytic vaccinia virus is administered only by intratumoral administration. In some embodiments, the immune checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraarterially and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic vaccinia virus comprises a heterologous nucleic acid sequence encoding a tumor antigen and optionally a cytokine, wherein the tumor antigen and optionally the cytokine are expressed in a cell infected with the virus, preferably a tumor cell.

In some embodiments, the invention provides a method of treating a tumor in a human, comprising co-administering to the human a combination comprising (a) a replicating oncolytic vaccinia virus and (b) an inhibitor of an immune checkpoint protein. In some embodiments of the method of treatment, the immune checkpoint protein is selected from PD-1, PD-L1, CTLA-4, LAG3, TIM3, and TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is CTLA-4. In some embodiments of the method of treatment, the immune checkpoint protein is PD-L1. In some embodiments of the method of treatment, the immune checkpoint protein is LAG3. In some embodiments of the method of treatment, the immune checkpoint protein is TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is PD-1. In some embodiments of the method of treatment, the immune checkpoint protein is TIM3. In some embodiments of the method of treatment, the tumor is a solid tumor. In some embodiments of the method of treatment, the tumor is colorectal cancer. In some embodiments of the method of treatment, the tumor is renal cell carcinoma. In some embodiments, the replicating oncolytic vaccinia virus is administered intratumorally, IV, and/or intraperitoneally. In some embodiments, the replicating oncolytic vaccinia virus is administered intratumorally. In some embodiments, the replicating oncolytic vaccinia virus is administered IV. In some embodiments, the replicating oncolytic vaccinia virus is administered intraperitoneally. In some embodiments, the replicating oncolytic vaccinia virus is administered intraarterially. In some embodiments, the replicating oncolytic vaccinia virus is administered exclusively intratumorally. In some embodiments, the immune checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intra-arterially and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic vaccinia virus comprises a heterologous nucleic acid sequence encoding a tumor antigen and optionally a cytokine, wherein the tumor antigen and optionally the cytokine are expressed in a cell infected with the virus, preferably a tumor cell.

In some embodiments, the invention provides a method of treating a tumor in a human, comprising co-administering to the human a combination comprising (a) a replicating oncolytic vaccinia virus, (b) an inhibitor of PD-1 and/or PD-L1, and (c) an inhibitor of an immune checkpoint protein. In some embodiments of the method of treatment, the immune checkpoint protein is selected from CTLA-4, LAG3, TIM3, and TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is CTLA-4. In some embodiments of the method of treatment, the immune checkpoint protein is LAG3. In some embodiments of the method of treatment, the immune checkpoint protein is TIGIT. In some embodiments of the method of treatment, the immune checkpoint protein is TIM3. In some embodiments of the method of treatment, the tumor is a solid tumor. In some embodiments of the method of treatment, the tumor is colorectal cancer. In some embodiments of the method of treatment, the tumor is renal cell carcinoma. In some embodiments, the replicating oncolytic vaccinia virus is administered by intratumoral, IV, and/or intraperitoneal administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by intratumoral administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by IV administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by intraperitoneal administration. In some embodiments, the replicating oncolytic vaccinia virus is administered solely by intratumoral administration. In some embodiments, the immune checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraarterially and the checkpoint inhibitor is administered systemically. In some embodiments, the replicative oncolytic vaccinia virus comprises a heterologous nucleic acid sequence encoding a tumor antigen and optionally a cytokine, wherein the tumor antigen and optionally the cytokine are expressed in a cell infected with the virus, preferably a tumor cell.

In some embodiments of the method of treatment, the tumor does not express an immune checkpoint protein or expresses a relatively low level of an immune checkpoint protein prior to administration of the replicating oncolytic vaccinia virus. In some embodiments, a high level is indicated by a tumor proportional score of greater than 50%. In some embodiments, a high level is indicated by a tumor proportional score of greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than about 99%, or about 100%. In some embodiments, a high level for a previously treated tumor is indicated by a tumor proportional score of greater than 1%. In some embodiments, a high level is indicated by PD-L1 expressed by greater than 50% of the tumor cells (e.g., greater than 50% of the tumor cells express PD-L1). In some embodiments, a high level is indicated by PD-L1 expressing greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, about 99%, or about 100% of the tumor cells. In some embodiments, a high level for a previously treated tumor is indicated by PD-L1 expressing greater than 1% of the tumor cells (e.g., greater than 1% of the tumor cells express PD-L1). In some embodiments, any PD-L1 diagnostic test can be used to measure PD-L1 expression. In some embodiments, when a PD-L1 checkpoint is measured, the PD-L1 DaKo Companion diagnostic test is used to measure PD-L1.

In some embodiments of the method of treatment, the method comprises a step of measuring the level of expression of the checkpoint protein in the tumor prior to administering the combination.

Administration of anticancer vaccinia virus and immune checkpoint inhibitors will follow the general protocol for administration of each specific therapy, taking into account the toxicity of the treatment, if any. It is expected that the treatment cycle will be repeated if necessary. It is also contemplated that in addition to the combination therapy of the present invention, various standard therapies as well as surgical interventions may be applied.

Treatment regimens can vary and often depend on the type of tumor, tumor location, disease progression, and the patient’s health and age. Certain types of tumors will require more aggressive treatment, but at the same time, certain patients may not be able to tolerate more rigorous protocols.

In certain embodiments, the tumor being treated may not be resectable, at least initially. Treatment with the combination therapy of the present invention may increase the resectability of the tumor, either due to shrinkage at the margins or by removal of certain specific invasive portions. After treatment, resection may be possible. Additional treatment following resection may serve to remove any residual disease at the tumor site.

Once a synergistic interaction between one or more components has been determined, the optimal range for effect and the absolute dose range of each component for effect can be definitively determined by dosing the components across different w/w ratios and doses for the patient requiring treatment. In humans, the complexity and expense of conducting clinical studies in patients renders this type of testing impractical as a primary model for synergy. However, observations of synergy in one species can be predictive of effects in another species, and animal models exist to measure synergy, as described herein, and the results of such studies can also be used to predict effective dose and plasma concentration ratio ranges and absolute doses, and the plasma concentrations required in other species by application of pharmacokinetic/pharmacodynamic methods. The established correlation between effects seen in tumor models and in humans suggests that synergy in animals can be demonstrated, for example, in human xenograft tumor models.

In some embodiments, the combination is used to treat and/or prevent cancer in a mammal. In some embodiments, the cancer includes, but is not limited to, brain cancer, head and neck cancer, esophagus cancer, skin cancer, lung cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, renal cell cancer, testicular cancer, rectal cancer, breast cancer and pancreatic cancer. In some embodiments, the cancer is selected from the group consisting of brain cancer, head and neck cancer, esophagus cancer, skin cancer, lung cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, renal cell cancer, testicular cancer, rectal cancer, breast cancer and pancreatic cancer. In a preferred embodiment, the combination is used to treat and/or prevent metastasis. In another preferred embodiment, the combination is used to treat cancers including but not limited to hepatocellular carcinoma, colorectal cancer, renal cell carcinoma, bladder cancer, lung cancer (including non-small cell lung cancer), gastric cancer, esophageal cancer, sarcoma, mesothelioma, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, uterine cervix and liver cancer. In some embodiments, the combination is used to treat cancers selected from the group consisting of hepatocellular carcinoma, colorectal cancer, renal cell carcinoma, bladder cancer, lung cancer (including non-small cell lung cancer), gastric cancer, esophageal cancer, sarcoma, mesothelioma, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, uterine cervix and liver cancer. In some embodiments, the combination is used to treat colorectal cancer, particularly metastatic colorectal cancer. In some embodiments, the mammal to be treated is a human. In another preferred aspect, the combination is used to treat a cancer that is resistant to one or more immune checkpoint inhibitors (e.g., the cancer is resistant to immunotherapy with PD-1, CTLA-4, LAG3 and/or TIGIT inhibitors). In some embodiments, the cancer is a solid tumor or a solid tumor.

The method comprises administering in combination a therapeutically effective amount of a replicating anti-cancer vaccinia virus and an immune checkpoint inhibitor. A therapeutically effective amount of the anti-cancer virus is defined as an amount sufficient to induce oncolysis - the disruption or lysis of cancer cells. Preferably, the anti-cancer vaccinia virus and the immune checkpoint inhibitor are administered in synergistically effective amounts. The term includes slowing, inhibiting, or reducing the growth or size of a tumor, and in certain instances, eradication of the tumor. In some embodiments, the effective amount of the anti-cancer vaccinia virus results in systemic dissemination of the therapeutic virus to the tumor, e.g., infection of a non-injected tumor. In some embodiments, the effective amount of the anti-cancer vaccinia virus is an amount sufficient to induce oncolysis - the disruption or lysis of cancer cells.

In some embodiments, the cancer treatment and/or prevention results in a reduction and/or shrinkage of tumor size and/or presence by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or about 100% after treatment. In some embodiments, the cancer treatment and/or prevention results in complete tumor regression after treatment. In some embodiments, the cancer treatment and/or prevention results in complete tumor remission after treatment. In some embodiments, the cancer is refractory or resistant to immune checkpoint inhibitor therapy. In some embodiments, the cancer is refractory or resistant to treatment with an anti-PD-1 antibody, an anti-PD-L1 antibody, and/or an anti-CTLA-4 antibody. In some embodiments, the cancer is resistant to treatment with an anti-PD-1 antibody. In some embodiments, the cancer is resistant to treatment with an anti-CTLA-4 antibody. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus and an immune checkpoint inhibitor. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, and/or TIM3. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus, an inhibitor of PD-1 or PD-L1, and an immune checkpoint inhibitor. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus, an inhibitor of PD-1 or PD-L1, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of CTLA-4, an inhibitor of LAG3, an inhibitor of TIGIT, or an inhibitor of TIM3. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus, an inhibitor of PD-1, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus, an inhibitor of PD-L1, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus, an inhibitor of PD-1, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of LAG3. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus, an inhibitor of PD-L1, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of LAG3. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus, an inhibitor of PD-1, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of TIGIT. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus, an inhibitor of PD-L1, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of TIGIT. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus, an inhibitor of PD-1, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of TIM3. In some embodiments, the treatment comprises administering a replicating oncolytic vaccinia virus, an inhibitor of PD-L1, and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an inhibitor of TIM3.

In some embodiments, the replicating oncolytic vaccinia virus is administered by intratumoral, IV, and/or intraperitoneal administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by intratumoral administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by IV administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by intraperitoneal administration. In some embodiments, the replicating oncolytic vaccinia virus is administered by intraarterial administration. In some embodiments, the replicating oncolytic vaccinia virus is administered exclusively by intratumoral administration. In some embodiments, the replicating oncolytic vaccinia virus is administered exclusively by intratumoral administration.

Checkpoint inhibitors as disclosed herein can be administered by a variety of routes, including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracerebrospinally, intraperitoneally, intrarectally, intracisternally, intratumorally, intravascularly, intradermally, or manually or by facilitated absorption through the skin, for example, using a skin patch or transdermal iontophoresis. In some embodiments, the checkpoint inhibitor is administered systemically. The checkpoint inhibitor can also be administered intravenously or intraarterially to the site of the pathologic condition, for example, to a blood vessel supplying the tumor. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, and/or TIM3.

In some embodiments, the replicating oncolytic virus is administered intratumorally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intravenously and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraperitoneally and the checkpoint inhibitor is administered systemically. In some embodiments, the replicating oncolytic virus is administered intraarterially and the checkpoint inhibitor is administered systemically.

In practicing the methods of the present invention, the total amount of the composition to be administered may be administered to the subject as a single dose, bolus, or by infusion over a relatively short period of time, or may be administered using a fractionated treatment protocol in which multiple doses are administered over an extended period of time. Those skilled in the art recognize that the amount of the composition to treat a pathological condition in a subject will depend on a number of factors, including the age and general health of the subject, as well as the route of administration and the number of treatments to be administered. Taking these factors into account, those skilled in the art will adjust the specific dosage, if necessary. Typically, the formulation of the composition and the route and frequency of administration are initially determined using Phase I and Phase II clinical trials.

In certain embodiments, the gate inhibitor is administered at a dose of 0.01 to 0.05 mg/kg, 0.05 to 0.1 mg/kg, 0.1 to 0.2 mg/kg, 0.2 to 0.3 mg/kg, 0.3 to 0.5 mg/kg, 0.5 to 0.7 mg/kg, 0.7 to 1 mg/kg, 1 to 2 mg/kg, 2 to 3 mg/kg, 3 to 4 mg/kg, 4 to 5 mg/kg, 5 to 6 mg/kg, 6 to 7 mg/kg, 7 to 8 mg/kg, 8 to 9 mg/kg, 9 to 10 mg/kg, at least 10 mg/kg or any combination thereof. Suitable dosages of the checkpoint inhibitor range from about 0.5 mg/kg to 25 mg/kg, preferably from about 1 mg/kg to about 20 mg/kg, more preferably from about 2 mg/kg to about 15 mg/kg. In certain embodiments, the checkpoint inhibitor is administered at least once per week, at least twice per week, at least three times per week, at least once every two weeks, or at least once per month or several months. In certain embodiments, the checkpoint inhibitor is administered as a single dose, in two doses, in three doses, in four doses, in five doses, or in six or more doses. Preferably, the checkpoint inhibitor is administered intravenously (e.g., by intravenous infusion or injection) or intratumorally. As a non-limiting example, ipilimumab is preferably administered by intravenous infusion at a dose of 3 mg/kg every three weeks for a total of four doses. In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, CTLA-4, LAG3, TIGIT and/or TIM3.

A. Additional anticancer treatments

One or more additional chemotherapeutic agents may be administered in any combination with the combinations of the present invention, including but not limited to, 5-fluorouracil (FU), folinic acid (FA) (or leucovorin), methotrexate, capecitabine (Xeloda; an oral prodrug of 5-FU), oxaliplatin (Eloxatin), bevacizumab (Avastin), cetuximab (Erbitux), and panitumumab (Vectibix). These agents may be administered according to known treatment protocols. Typically, the additional chemotherapeutic agents are administered intravenously, except for capecitabine, which is in oral form.

In another aspect, the methods of the present invention further comprise administering an additional cancer therapy, such as radiation therapy, hormonal therapy, surgery, and combinations thereof.

Radiotherapy includes, but is not limited to, the direct delivery of γ-rays, X-rays, and/or radioisotopes to tumor cells. Other forms of DNA damaging agents, such as microwaves and UV irradiation, are also contemplated. All of these agents have the potential to cause extensive damage to DNA, to DNA precursors, to DNA replication and repair, and to chromosome assembly and maintenance. The dosage range for X-rays ranges from daily doses of 50 to 200 roentgens for long periods of time (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. The dosage range for radioisotopes varies greatly and depends on the half-life of the isotope, the intensity and type of radiation emitted, and absorption by the neoplastic cells.

Approximately 60% of people with cancer undergo some type of surgery, including preventive, diagnostic or staging, radical and palliative surgery. Radical surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormone therapy, gene therapy and/or alternative therapies.

Radical surgery includes resection, where all or part of the cancerous tissue is physically removed, excised and/or destroyed. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electrosurgery and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with the removal of epidermal cancer, precancerous lesions or incidental amounts of normal tissue.

When a portion or all of a cancerous cell, tissue or tumor is removed, a hole may be formed in the body. Treatment may be achieved by perfusion of the area, direct injection or local application using additional anticancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments may likewise be at various dosages.

Other forms of treatment for use with the present method include hyperthermia, a procedure in which the patient's tissues are exposed to high temperatures (up to 106°F). External or internal heating devices may be involved in the application of local, regional, or systemic hyperthermia. Local hyperthermia involves the application of heat to a small area, such as a tumor. The heat may be generated externally by radiofrequency waves that target the tumor from a device outside the body. Internal heat may involve sterile probes, including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennas, or radiofrequency electrodes.

The patient's organ or limb is heated for local therapy, which is achieved by using a device that creates high energy, such as a magnet. Alternatively, some of the patient's blood may be removed and heated before being perfused to the area to be heated internally. Whole-body heating may also be performed if the cancer has spread throughout the body. For this purpose, warm blankets, hot wax, induction coils, and heat chambers may be used.

Hormone therapy may also be used in combination with the present invention or with any of the other cancer therapies described above. The use of hormones may be used in the treatment of certain cancers, such as breast, prostate, ovarian or cervical cancer, to lower the levels of or block the effects of certain hormones, such as testosterone or estrogen.

B. Compositions and Formulations

The replicating anti-cancer vaccinia virus of the pharmaceutical combination is administered directly to tumor cells to treat cancer and/or thereby, and the pharmaceutical compositions disclosed herein are formulated for the desired route of administration (e.g., by intratumoral injection, intravenously, intraarterially, and/or intraperitoneally). In some embodiments, the replicating anti-cancer vaccinia virus of the pharmaceutical combination is formulated for administration by intratumoral, intravenous, intraarterial, and/or intraperitoneal routes of administration. In some embodiments, the replicating anti-cancer vaccinia virus of the pharmaceutical combination is formulated for administration by intratumoral administration. In some embodiments, the replicating anti-cancer vaccinia virus of the pharmaceutical combination is formulated for administration by intravenous administration. In some embodiments, the replicating anti-cancer vaccinia virus of the pharmaceutical combination is formulated for administration by intraarterial administration. In some embodiments, the replicating anti-cancer vaccinia virus of the pharmaceutical combination is formulated for administration by intraperitoneal administration. In some embodiments, the replicative oncolytic vaccinia virus of the pharmaceutical combination is formulated for administration solely by intratumoral administration.

Intratumoral injection of the oncolytic vaccinia virus may be accomplished by any method used for the injection of a syringe or solution, provided that the expression construct can pass through a needle of a particular gauge required for injection. A novel needleless injection system having a nozzle defining an ampoule chamber for holding the solution and an energy device for forcing the solution out of the nozzle to the delivery site has recently been described (U.S. Pat. No. 5,846,233, incorporated herein by reference). Also described is a syringe system for use in gene therapy which allows multiple injections of predetermined amounts of solution at precisely arbitrary depths (U.S. Pat. No. 5,846,225, incorporated herein by reference).

Solutions of the active compound as the free base or a pharmaceutically acceptable salt may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (see, specifically, U.S. Pat. No. 5,466,468, which is incorporated herein by reference in its entirety). In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and/or vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable preparations can be brought about by the use of agents which delay absorption, for example, aluminum monostearate and gelatin.

For intratumoral injection in aqueous solutions, for example, the solution may be suitably buffered if necessary, and the liquid diluent provided isotonic with sufficient saline or glucose. In this regard, sterile aqueous media that may be used will be known to those skilled in the art in light of the present disclosure. For example, it may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed injection site (see, e.g., "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The individual responsible for administration will determine the appropriate dose for the individual subject in any event. In addition, for human administration, the formulation should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biopharmaceuticals.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, if required. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions disclosed herein may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, and the like. When formulated, the solution will be administered in a manner compatible with the dosage form and in a therapeutically effective amount. The formulation is readily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and preparations for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or preparation is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

The phrases "pharmaceutically-acceptable" or "pharmaceutically-acceptable" refer to molecular entities and compositions that do not produce allergic or similar untoward reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art. Typically, such compositions are prepared for injection as liquid solutions or suspensions; solid forms suitable for solution in liquid or suspension in liquid prior to injection may also be prepared.

Example

The following examples are provided for the purpose of illustrating various embodiments of the present invention and are not meant to limit the invention in any way. Those skilled in the art will readily recognize that the present invention is well suited to carrying out the objects and obtaining the ends, conclusions, and advantages mentioned, as well as the ends, conclusions, and advantages inherent in the present disclosure. As with the methods described herein, the examples represent presently preferred embodiments, are illustrative, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are included within the spirit of the invention as defined by the claims.

Example 1

The ability of combination therapy with anticancer vaccinia virus and checkpoint inhibitor(s) to suppress tumors in a synergistic mouse model of metastatic renal cell carcinoma (Renca) was evaluated. The growth pattern of this tumor closely mimics the growth pattern of human adult renal cell carcinoma, particularly with regard to spontaneous metastases to the lung and liver. The Renca model is hypervascular, immunoreactive, and resistant to anti-PD-1 immunotherapy. Since infection is independent of the tissue of origin, the Renca model is relevant for all cancers.

Materials and Methods

Mice and Cell Lines - Specific pathogen-free BALB/c male mice were housed under filter top cages with access to food and water on a 12-h light/dark cycle. All mice were anesthetized by intramuscular injection of an anesthetic combination (80 mg/kg ketamine and 12 mg/kg xylazine) prior to sacrifice. Renca renal cell carcinoma cell line and CT26 colon carcinoma cell line were obtained from ATCC and cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO ₂ in the presence of antibiotics.

Virus proliferation -

mJX594 is a Western Reserve vaccinia virus engineered to contain a disruption of the viral thymidine kinase gene and an insertion of murine GMCSF-GFP (mGMSCF-GFP) under the control of a synthetic early late promoter. HeLaS3 cells were infected with mJX594 at a multiplicity of infection (moi) of 1 to 3 at 100% confluence and incubated for 1.5 h at 37°C in a CO ₂ incubator, followed by application of DMEM containing 2.5% FBS and incubation for 48 to 72 h. Cells were harvested by centrifugation and the supernatant was discarded. Cells were resuspended in 10 mM Tris-Cl, pH 9.0, homogenized in a Dounce homogenizer, and centrifuged. The cell pellet was resuspended in 10 mM Tris-Cl, pH 9.0, centrifuged, and the supernatant was combined with the first supernatant. The supernatant was combined with the first supernatant. The sonicated lysate was then topped with 36% sucrose and centrifuged at 32,900×g for 80 min at 4°C. The pellet was then resuspended in 10 mM Tris-Cl, pH 9.0, and stored below -60°C.

ICI inhibitors (also referred to herein as immune checkpoint inhibitors) -

Antibodies to CTLA-4 and PD-1 were purchased from BioExcel. 9D9 monoclonal antibody reacts with mouse CTLA-4. Isotype: mouse IgG2b. J43 monoclonal antibody reacts with mouse PD-1. Isotype: Armenian hamster IgG.

Tumor Models and Treatment Schedules -

To generate a clinically relevant renal tumor model, a suspension of Renca tumor cells (renal cancer, isogenic) (5 × 10 ⁵ cells in 100 μl) was injected subcutaneously into the right dorsal flank of 8- to 10-week-old immunocompetent Balb/c male mice. When the mean tumor volume exceeded 50 mm3, mice were randomized and received the treatment regimens described below.

Tumor size was measured every 3 days in all groups using digital calipers.

Tumor volume was calculated according to the formula 0.5 × A × B2, where A is the largest diameter of the tumor and B is its perpendicular diameter. After the indicated days, mice were sacrificed by _CO2 , and tissues were collected for further analysis.

Histological analysis -

For immunofluorescence studies, samples were fixed in 1% PFA, dehydrated overnight in 20% sucrose solution, and embedded in tissue freezing medium (Leica). Frozen blocks were cut into 50 μm sections. Samples were blocked with 5% goat (or donkey) serum in PBST (0.03% Triton X-100 in PBS) and then incubated for 3 h at room temperature (RT) with the following primary antibodies: anti-GFP (rabbit, Millipore), anti-CD31 (hamster, clone 2H8, Millipore), anti-VEGFR2 (rabbit, Cell signaling), anti-CD8a (rat, BD Pharmingen), anti-CD11b (rat, BD Pharmingen), anti-FoxP3 (rat, eBioscience), anti-Caspase 3 (rabbit, R&D systems), anti-vaccinia (rabbit, Abcam), and anti-PD-L1 (rat, eBiosciences)). After several washes, the samples were incubated with the following secondary antibodies (FITC-, Cy3-, or Cy5-conjugated anti-hamster IgG (Jackson ImmunoResearch), FITC- or Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch), Cy3-conjugated anti-rat IgG (Jackson ImmunoResearch), or Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch)) for 2 h at room temperature. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen). The samples were then mounted with fluorescent mounting reagent (DAKO), and immunofluorescence images were acquired using a Zeiss LSM880 confocal microscope (Carl Zeiss).

Morphometric analysis -

Density measurements of blood vessels, CD8 T cells, or apoptotic areas were performed using ImageJ software (http://rsb.info.nih.gov/ij). CD31, CD8, or caspase3+ area/random 0.42㎟ area was measured in peritumoral and intratumoral areas. All measurements were performed on at least five different fields per mouse.

Flow cytometry -

The collected tumor tissues were minced and incubated in FACS buffer (1% FBS in PBS) containing collagenase D (Roche) and DNase I (Roche) at 37°C for 1–2 h in a shaking water bath. The lysed cells were filtered through a 40 μm nylon mesh to remove cell clumps. RBCs were removed by incubating the cell suspension in ACK lysis buffer at RT for 5 min. The harvested single cells were incubated for 30 minutes in FACS buffer with the following antibodies (PerCP-cy 5.5-conjugated anti-mouse CD45 (rat, eBioscience), APC-conjugated anti-mouse CD3e (hamster, eBioscience), FITC-conjugated anti-mouse CD4 (rat, eBioscience), PE-conjugated anti-CD8a (rat, eBioscience), FITC-conjugated anti-mouse CD11b (rat, eBioscience), APC-conjugated anti-mouse Gr1 (rat, eBioscience), and APC-conjugated anti-mouse CD11c (hamster, eBioscience)).

Statistical Analysis -

Values are presented as mean ± standard deviation. Statistical differences between means were determined by independent Student's t-test or one-way ANOVA followed by the Student-Newman-Keuls test. Statistical significance was set at p<0.05.

result

Antitumor efficacy of vaccinia virus + anti-PD-1 combination therapy

For combination therapy with anti-PD-1 and mJX-594, 5 × 10 ⁵ Renca tumor cells were injected into the right dorsal flank of BALB/c mice, and then treatment was initiated when the tumor size reached 50 to 100 mm3 (day 0). The mice were randomized into four treatment groups: (i) control group: PBS was injected intratumorally every 3 days; (ii) mJX-594 monotherapy group: 1 × 10 ⁷ pfu of mJX-594 was injected intratumorally every 2 days and then for a total of three times on days 0, 2, and 4; (iii) anti-PD-1 monotherapy group: 10 mg/kg of antibody was injected intraperitoneally every 3 days and then for a total of four times on days 0, 3, 6, and 9; (iv) mJX-594 + anti-PD-1 combination group: mJX-594 and anti-PD-1 were administered in parallel. mJX-594 was injected intratumorally every other day on days 0, 2, and 4 for a total of 3 injections, and anti-PD-1 was administered intraperitoneally on days 0, 2, 4, 6, and 9 for a total of 5 injections (see Figure 1A ).

Compared to the control group, tumor growth inhibition was observed in the mJX-594 monotherapy group and the mJX-594/anti-PD-1 combination group (“combination group”) (see Figure 2A ). The inhibition of tumor growth was more significant in the combination group (see Figure 2A comparing “PD1+mJX594” to “mJX594” and “PD1”). No tumor growth inhibition was observed in the anti-PD-1 monotherapy group (see Figure 2A comparing “PD1” to “control”). Tumor weight was found to be reduced in the mJX-594 monotherapy group (see Figure 2B ). A significant reduction in tumor weight was observed in the combination group, which was substantially greater than the reduction observed in the mJX-594 monotherapy group (see Figure 2B ). Thus, co-administration of mJX-594 and anti-PD-1 resulted in a significant reduction in tumor weight and volume compared to either monotherapy alone.

CD8 T-cell infiltration in both the peritumoral and intratumoral regions was increased in the PD-1 monotherapy, mJX-594 monotherapy, and combination therapy groups (see Figure 3A ). CD8 T-cell infiltration was increased in the peritumoral region than in the central region in the anti-PD-1 monotherapy group, whereas the mJX-594 group showed more CD8 T-cell infiltration in both the central and peripheral regions (see Figure 3B ). Combination administration of mJX-594 and anti-PD-1 antibody significantly increased intratumoral T-cell infiltration compared to the control group and compared to monotherapy with either agent alone, as measured by peritumoral and intratumoral CD8+ staining (see Figure 3B ). Reduced vascular density was also observed in the treatment group compared to the control group (see Figure 3B ).

In both the tumor periphery and tumor core, PD-L1 expression levels were increased in the anti-PD-1 monotherapy, mJX-594 monotherapy, and combination therapy groups compared to the control group (see Figure 4A ). The anti-PD-1 monotherapy group showed increased PD-L1 expression levels in the peripheral region more than in the core region, whereas the mJX-594 monotherapy group showed similar increases in PD-L1 expression levels in both the peripheral and core regions (see Figure 4A ). Co-administration of JX-595 and anti-PD-1 antibodies resulted in an increase in intratumoral PD-L1 expression compared to monotherapy with either antibody alone (see Figure 4A ). Renca tumors are resistant to anti-PD1 immunotherapy. The increase in tumor PD-L1 expression in the combination therapy group reflects the sensitization of these tumors to anti-PD-1 therapy, accompanied by infiltration of CD8+ T cells into the tumor, and is an indicator of therapeutic efficacy. These patterns suggest that T cells are immunosuppressed at baseline and unable to infiltrate the tumor microenvironment. mJX-594 induces inflammation and vasodilation, allowing T cells to exert antitumor effects. Co-administration of mJX-594 and anti-PD-1 antibodies results in activation and infiltration of T cells into the central tumor area.

Intratumoral apoptosis, as measured by caspase 3 staining, was observed to be increased in the PD-1 monotherapy, mJX-594 monotherapy, and combination therapy groups compared to the control group (see Figure 4B ). A significant increase in intratumoral apoptosis was observed in the combination therapy group compared to either monotherapy group (see Figure 4B ). The antiangiogenic effect combined with apoptosis in the combination therapy group (shown in Figure 3B ) suggests significant tumor necrosis in the combination therapy group.

Changes in the immune microenvironment were observed after concurrent mJX-594 and anti-PD-1 antibody compared to control and compared to monotherapy with either agent as measured by CD4 and CD11b (see Figure 5a ). Importantly, tumor infiltration of CD8 T cells was highest in the concurrent combination group. Depletion of CD8+ cells using anti-CD8 antibodies resulted in reduced tumor growth inhibition, confirming the role of these cells in the anti-tumor effect (data not shown). Myeloid-derived suppressor cells (MDSC) were increased in the mJX-594 and concurrent combination group compared to control. Traditionally, CD11b+Gr1+ cells have been simply considered as immunosuppressive cells; However, recent evidence has demonstrated that these cells cannot be so simply determined (in contrast to the relative increase in MDSC observed in the concurrent treatment group with αPD1, with a decrease in MDSC observed in the concurrent treatment group with αCTLA4) ( Figures 10A - B ).

The effect of sequential versus parallel co-administration of mJX-594 and PD-1 ± CTLA4 on tumor growth was evaluated. 5 × 10 ⁵ Renca (renal cancer, isogenic) cells were injected subcutaneously into the right flank of 8-week-old BALB/c immunocompetent mice. Treatment was initiated when tumors reached 50 mm3 in size (day 0).

Mice were separated into four treatment groups: (i) control group: PBS was injected on days 0, 3, 6, 9, 12, and 15; (ii) mJX-594 + anti-PD-1 sequential combination group: mJX-594 was injected intratumorally on days 0, 3, 6, and 9, and anti-PD-1 was injected intraperitoneally on days 6, 9, 12, and 15; (iii) mJX-594 and anti-PD-1 concurrent combination group: mJX-594 and anti-PD-1 were administered concurrently, mJX-594 was injected intratumorally on days 0, 3, 6, and 9, and then anti-PD-1 was administered intraperitoneally on days 0, 3, 6, 9, 12, and 15; (iv) mJX-594 + anti-PD-1 + anti-CTLA4 triple parallel combination group: mJX-594 was injected intratumorally on days 0, 3, 6, and 9; anti-PD-1 and CTLA4 were injected intraperitoneally on days 0, 3, 6, 9, 12, and 15 (see Figure 6 ).

Tumor growth was inhibited in the mJX-594 + anti-PD-1 sequential and concurrent administration groups as well as in the mJX-594 + anti-PD-1 + anti-CTLA4 triple concurrent administration group compared to the control group (see Figure 7 ). Surprisingly, concurrent administration of mJX-594 and anti-PD-1 resulted in a more significant inhibition (delay) of tumor growth than the sequential administration of these agents (see Figure 7 ). Additional delay in tumor growth was observed in the triple concurrent administration group (“Combination (mJX-594 + αPD1 + αCTLA4”)” (see Figure 7 ).

When the tumor size of each mouse was compared between treatment groups, tumor regression was observed in the combination group compared to the control group. While tumor regression was observed on day 12 in the sequential group, tumors tended to be suppressed on day 6 in the parallel and triple parallel combination groups.

These results surprisingly suggest that the dosing regimen, and potentially the route of administration, can significantly influence the anti-tumor efficacy of the combination therapy. In particular, when vaccinia virus and a checkpoint inhibitor are co-administered, vaccinia virus synergizes with the checkpoint inhibitor (anti-PD-1, CTLA-4) to induce a strong anti-tumor immune response. In some instances, a strong anti-tumor immune response was observed when vaccinia virus was administered intratumorally as part of the co-administration. The synergistic anti-tumor effect observed for the co-administration of vaccinia virus and a checkpoint inhibitor is particularly surprising since it is understood in the art that immune checkpoint inhibitors inhibit the replication of oncolytic viruses, such as vaccinia (Rojas et al., J. Immunol., 192(1 Supplement): 142.3 (2014)).

Vaccinia virus + anti-CTLA4

5× ¹⁰⁵ Renca (renal cancer) cells were injected subcutaneously into the right flank of 8-week-old BALB/c immunocompetent mice. Treatment was initiated when tumor sizes reached 50–100 mm3 (day 0).

Mice were randomized into five treatment groups: (i) control group: PBS was injected intratumorally on days 0, 3, 6, 9, 12, and 15; (ii) mJX-594 monotherapy group: 1 × 10 ⁷ pfu of mJX-594 was injected intratumorally on days 0, 3, 6, and 9; (iii) anti-CTLA4 monotherapy group: 4 mg/kg of antibody was injected intraperitoneally on days 0, 3, 6, 9, 12, and 15; (iv) mJX-594 + anti-CTLA4 sequential combination group: mJX-594 and anti-CTLA4 were administered sequentially, mJX-594 was injected intratumorally on days 0, 3, 6, and 9, and anti-CTLA4 was administered intraperitoneally on days 6, 9, 12, and 15; (v) mJX-594 and anti-CTLA4 simultaneous combination group: mJX-594 and anti-CTLA4 were administered sequentially, mJX-594 was injected intratumorally on days 0, 3, 6, and 9, and anti-CTLA4 was injected intraperitoneally on days 0, 3, 6, 9, 12, and 15 (see Figure 8).

Tumor size was measured every 3 days in all groups. When observation was performed by _CO2 , mice were sacrificed, tumors were taken, and flow cytometry analysis was performed (CD4+ and CD8+ tumor infiltrating lymphocytes (TIL), Gr1+/CD11b+ MDSC).

It was confirmed that tumor growth was inhibited in all treatment groups compared to the control group (see Figure 9 ). Significantly greater tumor growth inhibition was observed in the combination treatment group, and the greatest tumor growth inhibition was observed in the combination administration group (see Figure 9 ).

When the tumor size of each mouse was compared in the treatment groups, tumor inhibition was confirmed in the combination group compared to the monotherapy and control groups. Increased CD8 T-cell infiltration was observed in all treatment groups compared to the control group (see Fig. 10a ). MDSC levels increased in the mJX-594 monotherapy group compared to the control group, whereas there was no significant change in the sequential combination treatment group, and a decrease in MDSC levels was observed in the concurrent combination treatment group (see Fig. 10b ).

Co-administration of JX929 and immune checkpoint inhibitors

The antitumor activity of JX929 (6×10 ⁷ pfu) administered IT in combination with intraperitoneally administered PD-1 checkpoint inhibitors was tested in the mouse Renca model described above. JX929 is a Western Reserve strain vaccinia virus with a disruption of the viral TK and VGF genes (TK/VGF phenotype) and does not express GM-CSF.

Cell line -

Murine RENCA cells (ATCC) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and maintained at 37°C with 5% _CO2 .

In vivo studies -

Eight-week-old female BALB/c mice were injected intraperitoneally into the left renal margin with RENCA cells (2 × 10 ⁶ cells) in 100 μl of PBS. On day 10 post-transplant, Renca tumor-bearing mice (50 mm3 to 100 mm3 as visualized by an IVIS® Spectrum in vivo imaging system) were treated intraperitoneally (i.p.) with (i) PBS (control), (ii) vaccinia virus (JX-929) monotherapy (6 × 10 ⁷ PFU on days 10, 11, and 12 post-transplant for a total of three doses), (iii) anti-PD1 monotherapy (BioExcel, West Lebanon, NH, 100 μl) (on days 10, 11, and 12 post-transplant for a total of three doses), or (iv) concurrent JX929 plus anti-PD1 therapy (JX-929 was administered in the morning followed by ICI in the afternoon of the same day with a 9-hour interval on days 10, 11, and 12 post-transplant, respectively).

Mice were sacrificed 2 days after the final treatment for additional histological and flow cytometric analyses.

Flow cytometry -

Peripheral blood samples were collected, and red blood cells were lysed with RBC lysis buffer. Cells were washed in PBS containing 1% FBS and stained with monoclonal mouse anti-CD8, rabbit anti-CD4, and rabbit anti-CD3 antibodies (Santa Cruz Biotechnology, CA, USA). Cells were fixed with 4% paraformaldehyde and then incubated with FITC-conjugated goat anti-rabbit or goat APC-conjugated anti-mouse antibodies (Santa Cruz Biotechnology). For each sample, 10,000 cells were analyzed using a FACS Calibur instrument (BD Biosciences, CA, USA).

Histological analysis -

After anesthetizing the mice, vital organs including tumor-bearing kidney and lung were obtained and fixed in 10% neutralized formalin (BBC Biochemical, Washington, USA). Tissues were embedded in paraffin, and sections (4 μm thick) were stained with hematoxylin and eosin for basic histological analysis. For immunofluorescence and immunohistochemistry, sections were stained by standard methods using a mouse monoclonal antibody specific for CD8 (Santa Cruz Biotechnology, CA, USA). Sections were then incubated with FITC-conjugated goat anti-mouse antibody (Santa Cruz Biotechnology) for immunofluorescence or with Vectastain® Elite ABC-Peroxidase Kit (Vector Laboratories, CA, USA) and visualized by Vector SG (Vector Laboratories) for immunohistochemistry. The weight and volume of harvested tumors from each treatment group were measured and compared.

ELISpot Analysis -

IFNγ-secreting cells were assessed using the ELISpot Mouse IFNγ Kit (Mabtech, Cincinnati, OH) according to the manufacturer's protocol. Spleens were isolated and prepared as single-cell suspensions. Splenocytes were mixed with RENCA tumor cells or from mice infected with vaccinia virus at a ratio of 5:1 and incubated for 24 h at 37°C. The intensity of specific spots was analyzed using ImageJ software (NTH).

Statistical Analysis -

All values are presented as mean ± standard deviation (SD). Statistical analyses were performed using Instat 3 (GraphPad Software, California, USA). One-way analysis of variance (ANOVA) and Bonferroni post hoc paired comparison test were used.

mJX594 treatment induces expression of checkpoint proteins.

Balb/c mice bearing Renca tumors exceeding 50 mm2 were administered four intratumoral doses of mJX594 (1 × 10 ⁷ on days 0, 3, 6, and 9, respectively) or PBS control according to the treatment regimen shown in Figure 1C.

Levels of immune checkpoint proteins in tumors of control and mJX594-treated animals were measured on day 0 (before treatment) and day 12 after sacrifice of treated animals. Figure 13 depicts the fold-change in checkpoint proteins following treatment in mJX594-treated mice compared to control mice. As shown in Figure 13 , mJX594 treatment induces expression of checkpoint proteins including PD-1 (4-fold increase), PD-L1, PD-L2, CTLA4 (greater than 2-fold increase), LAG3, TIM3 (greater than 3-fold increase), and TIGIT (greater than 2-fold increase).

Treatment with replicating oncolytic vaccinia virus results in dynamic changes in the tumor immune microenvironment, including significant increases in the checkpoint proteins PD-1, PD-L1, CTLA-4, LAG3, TIM3 and TIGIT, thereby sensitizing tumors to blockade of each of these checkpoint proteins by their respective checkpoint inhibitors. Clinical trials have demonstrated that tumor-infiltrating cell and checkpoint protein (e.g., PD-L1) expression is indicative of therapeutic amenability to checkpoint inhibitors (e.g., anti-PD-L1 therapy), supporting the efficacy of the combination therapies described herein in patients with tumors that express particular checkpoint proteins as well as in patients with tumors that express low levels (or do not express) of particular checkpoint proteins.

Example 2

Tumor microenvironment remodeling by intratumoral anticancer vaccinia virus enhances immune checkpoint blockade efficacy

outline

Cancer immunotherapy is a powerful and sustainable treatment modality, but its clinical benefits are still not universal. In this specification, we used mJX-594, a targeted and GM-CSF-mediated oncolytic vaccinia virus (VV), as a combination antagonist to immune checkpoint inhibitors (ICIs) in mice with transplanted renal cell carcinoma, colon carcinoma, and spontaneous breast cancer. Intratumoral injection of VV induced a profound remodeling of the tumor microenvironment, transforming the tumor from a non-T cell-inflamed to a T cell-inflamed tumor with increased numbers of CD8 ⁺ T cells and enhanced effector functions. Furthermore, combination therapy of VV and ICI could induce tumor regression with improved survival and anti-metastatic effects. Our findings indicate that VV induces strong antitumor immunity in combination with ICIs to overcome immunotherapy resistance.

Introduction

Cancer immunotherapy with immune checkpoint inhibitors (ICIs) targeting PD-1 or CTLA-4 has demonstrated strong and sustainable therapeutic efficacy and has emerged as a new weapon in the war against cancer (Hegde et al., 2016; Topalian et al., 2015; Wolchok and Chan, 2014). However, the clinical efficacy of ICIs is limited to tumors with a T cell-inflamed tumor microenvironment (TME) (Gajewski, 2015; Topalian et al., 2016). In poorly immunogenic tumors with few tumor-infiltrating lymphocytes (TILs), the TME lacks the type I interferon signature and chemokines for T cell recruitment (Gajewski et al., 2013). Moreover, tumor vasculature and stromal components may pose barriers to intratumoral trafficking of T cells and their effector functions on tumor cells (De Palma and Jain, 2017; Rivera and Bergers, 2015; Sharma et al., 2017). Therefore, these non-T cell inflammatory tumors require additional therapeutic interventions to appropriately remodel the TME to render these tumors more sensitive to ICI therapy.

Oncolytic viruses (OVs) have been proposed as a novel class of anticancer therapies, and OVs with different scaffolds and transgenes are currently in clinical trials (Bell, 2014; Lichty et al., 2014). The success of OVs was initially attributed to their faster replication and enhanced antitumor capabilities over the past decade, but they are now beginning to be recognized as immunotherapeutic, as the most robust and durable responses following oncolytic virotherapy are coupled with the successful induction of antitumor immunity by tumor-specific effector and memory T cells (Bell, 2014; Chiocca and Rabkin, 2014; Thorne, 2014). Nevertheless, since the therapeutic efficacy of OVs is significantly hampered by the immunosuppressive TME, releasing the brakes of the immune system is crucial to maximizing the immunotherapeutic efficacy of OVs (Bell and Ilkow, 2017; Hou et al., 2016; Liu et al., 2017). Therefore, the combination of OV and ICI is a rational and interesting strategy to overcome the poorly immunogenic and immunosuppressive TME.

JX-594 (pexastimogene devacirepvec, Pexa-vec) is an oncogenic vaccinia virus (VV) engineered to express the immune-activating transgene GM-CSF and a disrupted viral thymidine kinase gene (Kirn and Thorne, 2009). JX-594 has demonstrated impressive antitumor activity with low toxicity in preclinical and clinical studies, making it one of the most feasible and promising OV platforms for clinical studies (Breitbach et al., 2011a; Cripe et al., 2015; Heo et al., 2013; Park et al., 2008). In addition to its antitumor and angiogenic activities, JX-594 is proposed to exhibit in situ tumor vaccination effects, as it can induce an adaptive immune response against tumor antigens for selective tumor lysis and subsequent additional tumor antigen release (Breitbach et al., 201 lb; Breitbach et al., 2015a). Although JX-594 is currently undergoing a phase III randomized clinical trial in advanced hepatocellular carcinoma (Abou-Alfa et al., 2016), only a few studies have characterized its immunomodulatory functions in the primary TME as well as in distant lesions after JX-594 treatment (Kim et al., 2018). Furthermore, the optimal combination of JX-594 with immunotherapeutics, such as ICIs, has not yet been pursued or proven.

In this specification, we thoroughly dissected the dynamic remodeling of TME with a mouse mutant of JX-594 (mJX-594, WR.TK mGM-CSF) and investigated its immunotherapeutic potential to provide a rational combination strategy with ICI in poorly immunogenic tumor models.

result

mJX-594 transforms immunosuppressive non-inflammatory tumors into inflammatory tumors

To determine the immunomodulatory potential of the oncolytic virus mJX-594, we examined transient changes in the tumor microenvironment in poorly immunogenic Renca tumors following a single mJX-594 injection. Levels of mJX-594 were already high on day 1 post-injection, peaked on day 3, and were barely detectable on day 7 ( Figures 33A and 33B ). In contrast, tumor vasculature showed the opposite response to virus levels; tumor vessel density was markedly reduced between days 1 and 3, but recovered on day 7 post-injection and thereafter ( Figures 33A and 33B ), indicating that mJX-594 induced robust but transient tumor vascular collapse. Interestingly, the CD8 ⁺ cytotoxic T cell population within the intratumoral area, which contains the most important aspect of antitumor immunity, began to increase markedly on day 5, was maximal on day 7, and remained dense for 2 weeks post-injection ( Figures 33A and 33B ), clearly demonstrating a clear and long-lasting transformation of an ainflammatory tumor into a T-cell-inflamed tumor by mJX-594. In comparison, CD11c ⁺ dendritic cells (DCs) were transiently present on day 3, but declined thereafter ( Figures 33A and 33B ). PD-L1 expression levels were minimal on day 0 and were upregulated following mJX-594 treatment ( Figures 1A and 1B ). Most interestingly, the timing of PD-L1 upregulation follows immediately after the massive influx of CD8 ⁺ TILs ( Figure 1C ), indicating activation of a negative feedback pathway that attempts to suppress T cell-mediated immunity. Most PD-L1 expressing cells were cytokeratin ⁺ tumor cells, and some CD11b ⁺ myeloid cells also expressed PD-L1, whereas T cells did not (Fig. 1D). Thus, mJX-594 becomes a potent and sustainable antitumor immune enhancer by recruiting cytotoxic CD8 ⁺ T cells to cold tumors, as well as a transient tumor vasculature disruptor.

To elucidate the cancer immune pathways regulated by mJX-594, we thoroughly analyzed the changes in the expression levels of 750 immune-related genes after mJX-594 monotherapy using the PanCancer immune profiling panel. The results showed remarkable differences in the gene-related immune signatures between control- and mJX-594-treated tumors ( Figure 33E ). Approximately 100 immune-regulatory genes showed statistically significant changes in their expression levels, including those implicated in activation of type I IFN signaling, DC maturation, and T cell activation ( Figure 33F ). In particular, we observed a generally higher expression of both inhibitory immune checkpoint molecules (including Pd-1, Pd-11, Ctla-4, and Lag-3) and agonistic immune checkpoint molecules (including Icos, Gitr, and Cd27) in the TME compared to the control ( Figure 33G ). Moreover, further analysis of the TME revealed an increase in Th1 and Th2 response-associated genes, suggesting immunomodulation by mJX-594 monotherapy ( Figure 33G ). We also found that several genes implicated in TME and myeloid cells were significantly upregulated ( Figure 33G ), particularly the increase in Nos2 and Cd86 expression, indicating polarization of myeloid cells toward M1 macrophages. These results indicate that mJX-594 induces long-term immune activation via dynamic changes in the TME to remodel non-inflammatory tumors into T cell-responsive inflammatory tumors capable of responding to immune checkpoint blockade.

mJX-594 is CD8 ⁺ It increases T cell infiltration into tumors and induces myeloid cell repolarization.

mJX-594-induced tumor growth delay was dose-dependent ( Figures 34A and 34B ). Concomitantly, mJX-594-induced increases in CD8 ⁺ T cell infiltration in both the peritumoral and intratumoral regions were also dose-dependent ( Figures 34C and 34D ). In fact, flow cytometric subpopulation analysis of the lymphoid cell compartment also indicated that mJX-594-induced increases in the absolute numbers of intratumoral CD8 ⁺ and CD4 ⁺ T cells were dose-dependent ( Figures 34E and 34G ) . Although CD4 ⁺ Foxp3 ⁺ CD25 ⁺ regulatory T cell numbers were also increased after triple administration of mJX-594 ( Figure 34H ), the ratio of CD8 ⁺ T cells to regulatory T cells was 5.3-fold higher compared to the cell ratio in the control treatment ( Figure 34I ), indicating an overall increase in T cell effector function in the TME with mJX-594 treatment. Additionally, the expression of costimulatory and T cell activation markers ICOS and granzyme B (GzB) was increased in CD8 ⁺ T cells after mJX-594 treatment ( Figure 34J ). Additional subpopulation analysis of the myeloid cell compartment revealed no significant changes in the CD11b ⁺ Gr1 ⁺ myeloid cell fraction in tumors treated with mJX-594 ( Figure 34K ). However, the CD11b ⁺ Ly6G ^- Ly6C ⁺ monocyte myeloid cell fraction was increased, whereas the CD11b ⁺ Ly6G ⁺ Ly6C ^int granulocyte myeloid cell fraction was decreased, indicating polarization of myeloid cells after mJX-594 administration ( Figures 34L and 34M ). These findings demonstrate that repeated mJX-594 administration enhances antitumor immunity, resulting in increased infiltration of activated T cells and repolarization of myeloid cells.

Intratumoral injection of mJX-594 elicits systemic and tumor-specific immune responses

To determine whether local injection of mJX-594 could induce a systemic immune response in non-injected distal tumors, we administered mJX-594 to the right tumor after implantation of Renca tumors in both lateral flanks. This treatment inhibited the growth of both right and left (contralateral, not injected, side) Renca tumors ( Figure 35A ). Similar to the inhibition of tumor growth on both sides, the infiltration of CD8 ⁺ T cells into the intratumoral area was increased 7.9- and 5.5-fold in both right and left Renca tumors ( Figures 35B and C ), suggesting that local mJX-594 virotherapy can robustly activate systemic antitumor immunity.

Next, to rule out the possibility of direct virus spread to distal tumors via systemic circulation after local virotherapy, we tested virus replication in the left, non-injected Renca tumor and found no detectable vaccinia virus in the left tumor ( Figure 41 ), indicating that the antitumor activity of mJX-594 was immune-mediated and not a result of systemic virus spread.

To assess whether the observed systemic immune responses were tumor-specific, we performed similar experiments using mice implanted with Renca tumors in the right flank and CT26 tumors in the left flank. Intratumoral treatment of the right, Renca tumor with mJX-594 significantly reduced the growth of the injected tumor, whereas the growth of the left, untreated CT26 tumor was unaffected ( Figure 35D ). Microscopic analysis provided consistent results that CD8+ T cells accumulated in Renca but not CT26 tumors ( Figures 35E and 35F ), indicating that mJX-594 virotherapy induced tumor-specific CD8 ⁺ T cell responses. Thus, these results suggest that local mJX-594 treatment can induce systemic antitumor immunity and even tumor-specific lymphocytic infiltration in distant tumors.

Antitumor immunity plays a critical role in the overall therapeutic efficacy of JX.

To determine whether components of the immune system are responsible for the therapeutic efficacy of mJX-594, we tested its effects on tumors in mice treated with neutralizing antibodies to CD8, CD4 or GM-CSF ( Figure 36A ). Specifically, depletion of either CD8 ⁺ or CD4 ⁺ T cells abrogated the effective inhibition of tumor growth by mJX-594 monotherapy ( Figures 36B and 36C ), highlighting the importance of immune-mediated mechanisms rather than direct tumor lysis in mJX-594-induced tumor inhibition. Most interestingly, depletion of CD4 ⁺ T cells upon mJX-594 injection also significantly reduced the intratumoral infiltration of CD8 ⁺ T cells ( Figure 36D ), implicating CD4 ⁺ T cells in the activation and recruitment of CD8 ⁺ T cells in the TME. However, depletion of CD8 ⁺ T cells did not significantly alter CD4 ⁺ T cell infiltration ( Figure 36E ), indicating that CD8 ⁺ T cells do not influence CD4 ⁺ T cells in the TME. These data indicate that intratumoral treatment with mJX-594 induces priming of CD8 ⁺ and CD4 ⁺ T cells, which can interact with each other to mediate antitumor immunity. Previous virotherapy based on herpes and vaccinia viruses utilized GM-CSF as an immune-activating transgene to recruit and activate antigen-presenting cells (APCs) that subsequently trigger T cell responses. However, the use of GM-CSF remains controversial due to its potential immunosuppressive role in tumor progression, such as inducing proliferation of myeloid-derived suppressor cells (MDSCs) (Thorne, 2014). Consequently, we studied whether GM-CSF is required for the therapeutic effect of mJX-594. Interestingly, depletion of GM-CSF abrogated the anti-tumor effect of mJX-594 and reduced both CD8 ⁺ and CD4 ⁺ T cell levels, suggesting that GM-CSF is important for the immunotherapeutic efficacy of mJX-594 ( Figures 36C to 36E ). Thus, both CD8 ⁺ and CD4 ⁺ T cells are essential mediators of the anti-tumor effect of mJX-594, and GM-CSF could provide the immunotherapeutic benefit.

Combination of mJX-594 and immune checkpoint blockade results in synergistic antitumor effects by enhanced infiltration of T lymphocytes into tumors

To overcome resistance to ICI monotherapy, we evaluated the benefit of combining mJX-594 with ICI. The combination of αPD-1 and mJX-594 reduced tumor growth by 70%, whereas individual αPD-1 and mJX-594 monotherapy delayed tumor growth by 22.8% and 44% at 12 days post-treatment ( Figures 37A and 37B ). In support of these findings, microscopic analysis confirmed a marked increase in CD8 ⁺ T cell recruitment in both peritumoral (18.8-fold) and intratumoral (21.4-fold) regions of combination-treated tumors compared to controls ( Figures 37C and 37D ). CD31 ⁺ tumor blood vessels were significantly reduced in these regions (1.8-fold and 2.6-fold, respectively; Figures 37C and 37D ). Additionally, more extensive tumor apoptosis was noted in tumors treated with combination therapy compared to controls (Figures 5C and 5D). PD-L1 expression was minimal in control tumors, but was highly upregulated following mJX-594 treatment ( Figures 37C and 37D ). These findings suggest that TME-induced PD-L1 expression is an adaptive negative feedback mechanism that dampens antitumor immunity following oncolytic virotherapy, thus providing a rationale for combination therapy of mJX-594 and PD-1/PD-L1 to block the potentiation of the immunotherapeutic effects of mJX-594 ( Figure 37E ).

Overall, these results suggest that combination therapy can overcome resistance to mJX594 or αPD-1 monotherapy through enhanced antitumor immunity by increasing CD8 ⁺ T cell infiltration.

Similarly, combination therapy with αCTLA-4 and mJX-594 was also synergistic. Tumor growth was modestly inhibited by either mJX-594 (42.0%) or αCTLA-4 (20.0%) monotherapy, but a stronger inhibition (57.6%) was observed with combination therapy ( Figures 42A and 42B ). Additionally, greater accumulation of CD8 + T cells was observed in both peritumoral (27.0-fold increase) and tumor center (26.4-fold increase) regions following combination therapy compared to controls ( Figures 42C and 42D ). Along with the increased levels of intratumoral CD8 ⁺ T cells, CD31 ⁺ blood vessels were also significantly disrupted in both peritumoral and tumor center regions compared to controls (2.1-fold and 3.8-fold decrease ^, respectively; Figures 42C and 42E ). Furthermore, flow cytometry analysis showed that intratumoral infiltration of CD8 ⁺ and CD4 ⁺ T cells was also increased by mJX-594 and αCTLA-4 combination therapy ( Figures 42F to 42H ).

Taken together, these results suggest that combination therapy using mJX-594 and ICI can overcome immunotherapy resistance in the immunosuppressed TME, resulting in synergistic antitumor effects.

The efficacy of combination immunotherapy with intratumoral mJX-594 and ICI is not significantly affected by treatment schedule.

Because ICI can negatively affect viral replication and cause premature clearance of OV, several studies have investigated the optimal schedule of treatment using a combination of systemic oncolytic virotherapy and ICI, and reported that some combination schedules could lead to antagonism of therapeutic efficacy (Liu et al., 2017; Rojas et al., 2015). However, no similar study testing local oncolytic virotherapy has been reported. To establish the optimal combination schedule for intratumoral mJX-594 and ICI, we compared: (1) concurrent administration of mJX-594 and ICI (Schedule I); (2) initiation of ICI 3 days after administration of mJX-594 (Schedule II); and (3) administration of mJX-594 3 days after initiation of ICI (Schedule III; Figure 38A ). All combination schedules delayed tumor growth by approximately 40% ( Figures 38B and 38C ). Likewise, the levels of tumor-infiltrating CD8 ⁺ and CD4 ⁺ T cells were increased by >8-fold and >4.0-fold, respectively, which also showed markedly increased levels of ICOS and GzB expression in CD8 ⁺ T cells ( Figures 38D to 38F ).

Similar to the combination therapy with mJX-594 and αPD-1, the combination of mJX-594 and αCTLA-4 inhibited tumor growth by approximately 40%, regardless of treatment schedule ( Figures 43A and 43B ). Furthermore, intratumoral infiltration of CD8 ⁺ and CD4 ⁺ T lymphocytes (>7-fold and >7-fold increase, respectively), as well as GzB and ICOS expression in CD8 ⁺ T cells, was greater, regardless of treatment schedule ( Figures 43C to 43E ).

In summary, combination therapy of intratumoral mJX-594 injection and systemic immune checkpoint blockade induced effective antitumor immune responses regardless of the treatment schedule, suggesting that intratumoral administration of mJX-594 is not significantly affected by varying the schedule of ICI administration.

The triple combination of mJX-594, αPD1 and αCTLA4 can induce complete tumor regression and provide long-term survival benefit in transplanted renal cell carcinoma.

Since the dual combination of mJX-594 and ICI did not induce complete tumor regression, we studied the triple combination therapy of mJX-594, αPD-1, and αCTLA-4. The dual combination of αPD-1 and αCTLA-4 delayed tumor growth by 14.5%, mJX-594 monotherapy inhibited tumor growth by 36.9%, and the triple combination inhibited tumor growth by 76.5% ( Figures 39A and 39B ). Notably, a small minority (approximately 40%) of this triple combination group resulted in complete tumor regression, which was not observed in any other group ( Figure 39C ).

To determine whether the strong antitumor effects induced by the triple combination therapy could be translated into long-term survival benefits, we performed survival analyses of tumor-bearing mice. Indeed, mice treated with the triple combination therapy exhibited a remarkable survival benefit compared to the other treatments ( Figure 39D ). Furthermore, mice with complete tumor regression remained tumor-free for more than 12 weeks after the end of treatment and were completely protected against rechallenge with tumor cells, suggesting the establishment of effective, long-term immune memory ( Figure 39E ).

These findings demonstrate that triple combination immunotherapy has the potential to induce complete tumor regression and long-term survival.

Triple combination therapy induced antitumor immune responses in a spontaneous breast cancer model. Improve

To definitively demonstrate the long-term immunotherapeutic efficacy of the triple combination therapy in immune-resistant tumors, we used the MMTV-PyMT transgenic mouse model, a spontaneous breast cancer model with intrinsic resistance to cancer immunotherapy (Schmittnaegel et al., 2017). After 4 weeks of treatment, mice treated with the triple combination of mJX-594, αPD-1, and αCTLA-4 exhibited a significant reduction in overall tumor burden (by 38.7%) and in the number of palpable mammary tumor nodules compared to control mice ( Figures 40A - D ). Furthermore, the triple combination therapy resulted in a 48.1% reduction in the average size of individual tumor nodules and improved overall survival compared to the other treatments ( Figures 40E - F ). Histological analysis ( Figure 40G , see legend for detailed description) showed less invasive carcinomas with well-preserved tumor margins in the triple combination group, indicating that the triple combination effectively delayed tumor progression and invasion. In contrast, tumors treated with the dual combination of αPD-1 and αCTLA-4 exhibited an invasive carcinoma phenotype similar to the control group, infiltrating surrounding tissues and forming solid sheets of tumor cells. Accordingly, after triple combination therapy, intratumoral recruitment of CD8 ⁺ T cells was significantly increased by >50-fold compared to any other treatment ( Figures 40H and 40L ). However, tumor vascular density was similar between treatment groups ( Figure 40J ). These findings indicate that enhanced antitumor immunity, rather than vasoconstrictive effects, is important for the long-term therapeutic efficacy of triple combination immunotherapy with mJX-594 and ICI. Finally, the number of hematogenous lung metastases was significantly reduced in the triple combination group ( Figures 40K and 40L ), indicating an effective anti-metastatic effect of the triple combination therapy.

Taken together, these results demonstrate that triple combination immunotherapy with mJX-594 and ICI can induce robust antitumor immune responses even in poorly immunogenic spontaneous breast cancer models.

argument

In this specification, we demonstrate that combination therapy with mJX-594 and ICI is an effective therapeutic strategy for immune-resistant tumors. Combination therapy induces an immunological “boiling point” where cold, non-inflamed tumors become sufficiently inflamed to allow the host immune system to eradicate the tumor cells. The most pronounced effect was observed with triple immunotherapy with mJX-594, anti-PD-1 and anti-CTLA4, which induced complete regression in approximately 40% of Renca tumors, one of the most resistant isogenic to immunotherapy. This strong synergy can be explained by the complementary cooperation of OV and ICI.

JX-594 is the most advanced OV in clinical trials, which is known to act via multiple mechanisms (Abou-Alfa et al., 2016). Although it can rapidly induce direct tumor lysis and vascular collapse in tumors, these effects are transient and largely disappear within 1 week of injection. Subsequently, CD8 ⁺ T cells extensively infiltrate the tumor, initiating an antitumor immune response. However, at the same time, the tumor begins to evolve to avoid immune-mediated elimination by upregulating immune inhibitory checkpoint molecules such as PD-1, PD-L1 or CTLA-4 in the TME. Since the strongest and most sustainable antitumor effects of OVs are achieved when combined with successful induction and maintenance of antitumor immunity, it may be reasonable to combine ICIs with OVs to prevent premature shutdown of OV-induced antitumor immunity.

Although ICI monotherapy has revolutionized the cancer treatment landscape, its dramatic therapeutic responses have been limited to a subset of patients. This has given rise to the concept of immunologically ‘hot’ or ‘cold’ tumors; hot tumors respond well to ICIs because they are not immunologically depleted by TILs and have high expression of PD-L1, whereas cold tumors are refractory to ICIs due to the lack of CD8 ⁺ TILs and immunosuppressive TME (Bell and Ilkow, 2017; Gajewski et al., 2013). Therefore, current efforts focus on overcoming resistance to ICIs by converting immunologically cold tumors into hot tumors. In this context, our results suggest mJX-594 as an ideal combination state for ICIs, which can selectively replicate in tumor cells, destroy them, and release tumor antigens to stimulate the host immune system. Moreover, our study shows that TME from cold tumors can be dramatically shifted to hot state by inducing intratumoral inflammatory response (induction of Th1 response, activation and recruitment of T cells, upregulation of PD-L1, and polarization of myeloid cells toward antitumor activity). Interestingly, replication and spread of OVs are known to be more active in cold tumors with few immune cells to clear OVs, whereas hot tumors with ampules of TILs could induce early clearance of OVs and attenuate their therapeutic effects (Bell and Ilkow, 2017). Therefore, together with the results of this study, mJX-594 is an optimal combination mate for non-inflammatory cold tumors with inherent resistance to ICIs, especially to immunotherapy.

GM-CSF is the most commonly used genetic payload of OVs. The two most advanced OVs in clinical trials, T-Vec and Pexa-Vec (JX-594), both have GM-CSF arms. GM-CSF is generally known to induce the proliferation of various immune cells, such as DCs, but has the concern of unwanted proliferation of immunosuppressive cells, such as MDSCs (Hou et al., 2016). In the present study, we showed that mJX-594 did not significantly alter the fraction of intratumoral CD11b ⁺ Gr1 ⁺ cells. In addition, neutralization of GM-CSF abolished the therapeutic efficacy of mJX-594, which was partly due to the reduction of CD8 ⁺ TILs, indicating that GM-CSF plays an essential role in mJX-594-induced tumor immunity.

Previous studies have reported that the combination of OV and ICI induces an impressive immune response, but its therapeutic efficacy can be significantly influenced by the route of administration and treatment schedule. In particular, when both OV and ICI are administered systemically simultaneously, the combination can be antagonistic due to ICI-induced anti-viral immunity that may facilitate early viral clearance, indicating the importance of an appropriate time interval between OV treatments in inducing successful anti-tumor immunity. In the present study, local injection of mJX-594 consistently induced anti-tumor immunity without being significantly influenced by the administration sequence. We speculate that this is because intratumoral injection provides OV with sufficient time to induce inflammation in the TME before it is cleared by systemic anti-viral immunity. Therefore, in designing clinical trials of ICI and OV combinations, intratumoral OV therapy may be more feasible than systemic OV therapy with respect to the administration schedule.

In addition to our promising results utilizing the combination of mJX-594 and ICI, several clinical trials are already underway to investigate the efficacy of JX-594 in combination with αPD-1, αCTLA-4 or αPD-L1 to target various solid tumors including liver cancer, renal cell carcinoma and colon cancer (ClinicalTrials.gov: NCT03071094, NCT02977156, NCT03294083 and NCT03206073). Therefore, we expect to be able to validate our findings in a clinical setting in the near future.

In conclusion, this study demonstrated that intratumoral injection of mJX-954 induced a marked remodeling of the TME from a cold state to a hot state, and combined with ICI induced strong antitumor immunity, thereby overcoming immunotherapy resistance.

Experimental Procedure

Mice and cell lines

Male BALB/c mice, 6 to 8 weeks of age, were purchased from Orient Bio Inc. (Seongnam, Gyeonggi-do, South Korea) and female MMTV-PyMT transgenic mice (FVB/N) were purchased from Jackson Laboratory (Bar Harbor, MD, USA #002374). Mice were housed in a pathogen-free animal facility at CHA University (Seongnam, Gyeonggi-do, South Korea). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC, #170025) of CHA University and were performed according to the approved protocol. Renca murine renal cell carcinoma cell line and CT26 murine colon carcinoma cell line were obtained from the National Collection of Microorganisms (Manassas, VA, USA #CRL-2947) and the Korean Cell Line Bank (Seoul, Republic of Korea #80009). These cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium or Dulbecco's Modified Eagle Medium (DMEM), each supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and incubated at 37°C with 5% CO ₂ in an incubator.

Generation and quantification of viruses

mJX-594, provided by Sillajen, Inc. (Seoul, Korea), is a Western Reserve (WR) strain of vaccinia virus encoding murine GM-CSF at the vaccinia thymidine kinase gene locus under the control of the p7.5 promoter and was used throughout this study. The virus was amplified in HeLaS3 cells prior to purification. Briefly, HeLaS3 cells were infected with the recombinant vaccinia virus for 3 days, harvested by centrifugation, then homogenized and centrifuged once more. The virus-containing supernatant was layered onto a 36% sucrose cushion, centrifuged at 32,900 g, and the purified virus pellet was resuspended in 1 mM Tris, pH 9.0. To determine virus titers, serially diluted virus in serum-free DMEM was plated onto monolayers of U-2 OS cells for 2 h, followed by addition of 1.5% carboxymethylcellulose in DMEM supplemented with 2% FBS. After 72 h, cells were stained with 0.1% crystal violet, and plaques were counted.

Tumor models and treatments

Tumors were implanted into the right flank of wild-type BALB/c mice by subcutaneous injection of 2 × 10 ⁵ Renca cells. When tumors reached >50 mm 3, mice were treated with PBS or 1 × 10 ⁷ plaque forming units (pfu) of mJX-594 by intratumoral injection every 3 days. For both tumor models, 2 × 10 ⁵ Renca cells were injected subcutaneously into the right flank, and 1 × 10 ⁵ Renca or CT26 cells were implanted subcutaneously into the left flank 4 days later. For cell depletion studies, antibodies to CD4 (200 μg, clone GK1.5, BioExcel), CD8 (200 μg, clone 53-6.72, BioExcel), or GM-CSF (200 μg, clone MP1-22E9, BioExcel) were injected intraperitoneally together with mJX-594. For immune checkpoint blockade, anti-PD-1 (10 mg/kg, clone J43, BioExcel) and/or anti-CTLA-4 (4 mg/kg, clone 9D9, BioExcel) antibodies were injected intraperitoneally every 3 days with or without mJX-594 according to the dosing schedule. Tumors were measured every 2 or 3 days using digital calipers, and tumor volumes were calculated using the modified ellipsoid formula (1/2 × (length × width ² )). On day 50, surviving mice with complete tumor regression were rechallenged with 2 × 10 ⁵ Renca cells in the left flank and monitored for tumor growth and survival. Mice were euthanized when tumors reached 1.5 cm in diameter or when they became moribund.

Female MMTV-PyMT transgenic mice were purchased from Jackson Laboratories. At 9 weeks of age, the volume of all palpable tumor nodules (>20 mm3) was measured, and the total volume of all combined tumors was used to calculate tumor burden per mouse. MMTV-PyMT mice were randomized according to their initial tumor burden and treated with 1 × 10 7 ^pfu in the presence or absence of immune checkpoint inhibitors PD-1 (10 mg/kg) or CTLA-4 (4 mg/kg) at the indicated time points. After 4 weeks of treatment, mice were anesthetized and tissues were harvested for further analysis. Analyses for MMTV-PyMT were performed as previously described (Kim et al., 2014; Park et al., 2016).

Histological analysis

For hematoxylin and eosin (H&E) staining, tumors were fixed overnight in 4% paraformaldehyde (PFA). After tissue processing using standard procedures, samples were embedded in paraffin, cut into 3 μm sections, and stained with H&E. Immunofluorescence was performed on frozen tissue sections. Tumors were fixed in 1% PFA at room temperature, rinsed several times with PBS, permeabilized with 30% sucrose, and frozen in OCT compound. Cryosections (50 μm thick) were blocked with 5% normal goat serum in PBS-T (0.1% Triton X-100 in PBS) and then incubated overnight with the following primary antibodies: anti-vaccinia virus (rabbit, Abcam), anti-CD31 (hamster, clone 2H8, Millipore; rabbit, Abcam), anti-CD8 (rat, clone 53-6.7, BD Pharmogen), anti-CD11c (hamster, clone HL3, BD Pharmogen), anti-PD-L1 (rabbit, clone 28-8, Abcam), anti-caspase 3 (rabbit, R&D Systems), anti-Pan-cytokeratin (mouse, clone AE1/AE3, DAKO), anti-CD11b (rat, clone M1/70, BD Pharmogen), or anti-CD3e (hamster, clone 145-2C11, BD Pharmogen). After several washes, the samples were incubated for 2 h at room temperature with the following secondary antibodies: FITC-, Cy3- or Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch), FITC-conjugated anti-rat IgG (Jackson ImmunoResearch), FITC- or Cy3-conjugated anti-hamster IgG (Jackson ImmunoResearch), or FITC-conjugated anti-mouse IgG (Jackson ImmunoResearch). Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAP I, Invitrogen). Finally, the samples were mounted with fluorescent mounting agent (DAKO) and images were acquired with a Zeiss LSM 880 microscope (Carl Zeiss).

Morphometric analysis

Measurements of vaccinia virus, blood vessel, T lymphocyte and dendritic cell density, as well as measurements of myeloid cell immunity were performed using ImageJ software (http://rsb.info.nih.gov/ij). To determine the level of vaccinia virus infection, the VV ⁺ area was calculated per random 0.49 mm2 field in tumor sections. For blood vessel density, the CD31 ⁺ area per random 0.49 mm2 field was calculated in the peritumoral and intratumoral regions. The extent of cytotoxic T lymphocyte infiltration was expressed as the percentage of CD8 ⁺ area per 0.49 mm2 field in the peritumoral and intratumoral regions. Dendritic cell levels were measured by calculating the percentage of CD11c ⁺ area in random 0.49 mm2 fields. The extent of apoptosis was expressed as the percentage of caspase 3 ⁺ area in random 0.49 mm2 fields. To determine PD-L1 ⁺ cells, colocalization of PD-L1 ⁺ with Pan-CK +, CD11b ⁺ , and CD3 ⁺ was identified in random 0.01㎟ fields. Lung metastases in MMTV-PyMT mice were quantified by measuring tumor colonies >100 ㎛ in diameter. All measurements were performed in at least 5 fields per mouse.

Flow cytometric analysis of tumor-infiltrating immune cells

Tumors from each treatment group were minced and incubated with shaking at 37°C for 1 h in the presence of collagenase D (20 mg/mL, Roche) and DNase I (2 mg/mL, Roche). Cell suspensions were generated by repeated pipetting, then filtered through a 70 μm cell strainer, and lysed to remove red blood cells. After washing with PBS, resuspended cells were filtered through a nylon mesh. Single-cell suspensions from tumor tissues were blocked with antibodies to CD16/32 (clone 2.4G2, BD Pharmigene) and stained with a fixable viability dye (eFlouor450, eBioscience) to distinguish viable cells. For analysis of surface markers, cells were stained in PBS containing 1% FBS with antibodies targeting CD45 (30-F11, BD Pharmogen), CD4 (RM4-5, BD Pharmogen), CD8 (53-6.7, BD Pharmogen), CD3 (17A2 or 145-2C11, eBioscience), ICOS (7E.17G9 or 15F9, eBioscience), CD11b (M1/70, BD Pharmogen), F4/80 (BM8, eBioscience), MHC II (M5/114.15.2, eBioscience), Ly6C (HK1.4, eBioscience), Ly6G (1A8-Ly6g or RB6-8C5, eBioscience), or CD206 (MR5D3, eBioscience) for 30 min on ice. Cells were further permeabilized using a FoxP3 Fixation and Permeabilization Kit (eBioscience) and stained for FoxP3 (FJK-16, eBioscience), CD25 (PC61.5, eBioscience), or granzyme B (NGZB, eBioscience). Labeled cells were acquired using a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

RNA isolation and nanostring gene expression analysis

Total RNA was extracted from whole tumor lysates using TRIzol (Invitrogen), purified with ethanol; and RNA quality was confirmed using a Fragment Analyzer instrument (Advanced Analytical Technologies, Iowa, USA). Immune profiling was performed using a digital multiplex NanoString nCounter PanCancer Immune Profiling mouse panel (NanoString Technologies) using 100 ng of total RNA isolated from tumor tissue. Hybridization was performed at 65°C for 16-30 h by combining 5 μl of each RNA sample with 8 μl of nCounter Reporter and 2 μl of nCounter Capture Probe in hybridization buffer in a total reaction volume of 15 μl. Excess probe was removed by a two-step magnetic bead-based purification using the nCounter Prep Station (Nanostring Technologies). Specific target molecule abundance was quantified by counting individual fluorescent barcodes and evaluating the corresponding target molecules using the nCounter digital analyzer. For each assay, a high-density scan containing 280 fields of view was performed. Images of the immobilized fluorescent reporters in the sample cartridge were acquired with a CCD camera, after which data were collected using the nCounter digital analyzer. Data analysis was performed using nSolver software (Nanostring Technologies). mRNA profiling data were normalized to housekeeping genes and analyzed using R software (www.r-project.org).

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA) and PASW statistics 18 (SPSS). Unless otherwise indicated, values are expressed as mean +/- standard error of the mean (SEM).

The independent sample Student's t-test was used to test the statistical differences between means. The Kaplan-Meier method was used to generate survival curves, and the log-rank test was used to analyze the statistical differences between curves. The statistical significance level was set at p < 0.05.

References

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications may be made to the compositions and/or methods described herein and to the steps or sequence of steps of the methods without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

The examples set forth above provide those skilled in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes of carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference were individually incorporated in its entirety.

All headings and section designations are used for clarity and reference purposes only and should not be considered limiting in any way. For example, those skilled in the art will recognize the utility of combining various aspects from different headings and sections, where appropriate, while remaining consistent with the spirit and scope of the invention as described herein.

All references cited in this specification are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

As will be apparent to those skilled in the art, many modifications and variations of the present application can be made without departing from the spirit and scope thereof. The specific embodiments and examples described herein are provided by way of example only, and the present application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims (65)

A method for treating and/or preventing cancer in a subject in need of cancer treatment, comprising concurrently administering to the subject an effective amount of a combination comprising (a) a replicative oncolytic vaccinia virus and (b) one or more immune checkpoint inhibitors. A method for treating and/or preventing cancer, wherein the replicative anti-cancer vaccinia virus in claim 1 is administered intratumorally, intravenously, intraarterially or intraperitoneally. A method for treating and/or preventing cancer, wherein the replicative anti-cancer vaccinia virus in claim 2 is administered intratumorally. A method for treating and/or preventing cancer according to any one of claims 1 to 3, wherein the replicative anti-cancer vaccinia virus is administered in an amount effective to induce expression of an immune checkpoint protein in the tumor. A method for treating and/or preventing cancer according to any one of claims 1 to 4, wherein the tumor does not express the immune checkpoint protein or expresses the immune checkpoint protein at a relatively low level prior to administration of the replicative anti-cancer vaccinia virus. A method for treating and/or preventing cancer according to any one of claims 1 to 5, wherein the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to the immune checkpoint protein, preferably a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein or a combination thereof. A method for treating and/or preventing cancer according to any one of claims 1 to 6, wherein the checkpoint inhibitor inhibits an immune checkpoint protein selected from the group consisting of cytotoxic T-lymphocyte antigen-4 (CTLA4), programmed cell death protein 1 (PD-1), PD-L1, PD-L2, B7-H3, B7-H4, herpesvirus entry mediator (HVEM), T-cell membrane protein 3 (TIM3), galectin 9 (GAL9), lymphocyte activation gene 3 (LAG3), V-domain immunoglobulin (Ig)-containing inhibitor of T-cell activation (VISTA), killer-cell immunoglobulin-like receptor (KIR), B and T lymphocyte attenuator (BTLA), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), indoleamine 2,3-dioxygenase (IDO), and combinations thereof. A method for treating and/or preventing cancer, wherein the checkpoint inhibitor in claim 7 is a monoclonal antibody that selectively binds to PD-1 or PD-L1, preferably selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab. A method for treating and/or preventing cancer, wherein in claim 7, the checkpoint inhibitor is a monoclonal antibody that selectively binds to CTLA4, preferably selected from the group consisting of ipilimumab and tremelimumab. A method for treating and/or preventing cancer according to any one of claims 1 to 9, wherein a multiple checkpoint inhibitor is administered to the subject in combination with the anticancer vaccinia virus. In the 10th paragraph, to the subject,
(a) CTLA4 inhibitors and PD-1 inhibitors and replicative oncolytic vaccinia virus;
(b) CTLA4 inhibitors and IDO inhibitors and replicative oncolytic vaccinia virus;
(c) PD-1 inhibitor and IDO inhibitor and replicative anticancer vaccinia virus; or
(d) PD-1 inhibitors, CTLA4 inhibitors, IDO inhibitors, and replicative oncolytic vaccinia virus;
(e) LAG3 inhibitor and PD-1 inhibitor and replicative anticancer vaccinia virus; or
(f) TIGIT inhibitor and PD-1 inhibitor and replicative anticancer vaccinia virus
A method for treating and/or preventing cancer, comprising administering concurrently. A method for treating and/or preventing cancer according to any one of claims 1 to 11, wherein the replicative anti-cancer vaccinia virus is a Wyeth strain, a Western Reserve strain, a Lister strain or a Copenhagen strain. A method for treating and/or preventing cancer according to any one of claims 1 to 12, wherein the vaccinia virus comprises one or more genetic modifications to increase the susceptibility of the virus to cancer cells, preferably wherein the virus is engineered to lack a functional thymidine kinase and/or to lack a functional vaccinia growth factor. A method for treating and/or preventing cancer according to any one of claims 1 to 13, wherein the vaccinia virus comprises a functional 14L and/or F4L gene. In any one of claims 1 to 14, the vaccinia virus (i) preferably a cytokine selected from GM-CSF, IL-2, IL-4, IL-5 IL-7, IL-12, IL-15, IL-18, IL-21, IL-24, IFN-γ, TNF-α, and/or (ii) preferably BAGE, GAGE-1, GAGE-2, CEA, AIM2, CDK4, BMI1, COX-2, MUM-1, MUC-1, TRP-1 TRP-2, GP100, EGFRvIII, EZH2, LICAM, Livin, Livinβ, MRP-3, Nestin, OLIG2, SOX2, Human Papillomavirus-E6, Human Papillomavirus-E7, ART1, ART4, SART1, SART2, SART3, B-cyclin, β-catenin, Gli1, Cav-1, Cathepsin B, CD74, E-cadherin, EphA2/Eck, Fra-1/Fosl 1, ganglioside/GD2, GnT-V, β1,6-N, Her2/neu, Ki67, Ku70/80, IL-13Ra2, MAGE-1, MAGE-3, NY-ESO-1, MART-1, PROX1, PSCA, SOX10, SOX11, survivin, caspase-8, UPAR, CA-125, PSA, p185HER2, CD5, IL-2R, Fap-α, tenascin, melanoma-associated antigen p97 and WT-1, regulator of G-protein signaling 5 (RGS5), survivin (BIRC5 = baculovirus inhibitor of apoptosis repeat-containing 5), insulin-like growth factor-binding protein Intermediate growth factor-binding protein 3 (IGF-BP3), thymidylate synthase (TYMS), hypoxia-inducible protein 2, hypoxia-inducible fat granule-associated (HIG2), matrix metallopeptidase 7 (MMP7), Prune homolog 2 (PRUNE2), RecQ protein-like (DNA helicase Q1-like) (RECQL), leptin receptor (LEPR), ERBB receptor feedback inhibitor 1 (ERRFI1), lysosomal protein transmembrane 4 alpha (LAPTM4A); A method for treating and/or preventing cancer, wherein the cell is engineered to express a tumor antigen selected from RAB1B, RAS oncogene family (RABIB), CD24, Homo sapiens thymosin beta 4, X-linked (TMSB4X), Homo sapiens S100 calcium binding protein A6 (S100A6), Homo sapiens adenosine A2 receptor (ADORA2B), chromosome 16 open reading frame 61 (C16orf61), ROD1 regulator of differentiation 1 (ROD1), NAD-dependent deacetylase sirtuin-2 (SIR2L), tubulin alpha 1c (TUBA1C), ATPase inhibitor factor 1 (ATPIF1), substrate antigen 2 (STAG2), nuclear casein kinase and cyclin-dependent substrate 1 (NUCKS1). A method for treating and/or preventing cancer according to any one of claims 1 to 15, wherein the vaccinia virus is administered in an amount of about 10 ⁷ to about 10 ¹¹ pfu, preferably about 10 ⁸ to 10 ¹⁰ pfu, more preferably about 10 ⁹ to 10 ¹⁰ pfu. A method for treating and/or preventing cancer according to any one of claims 1 to 16, wherein the checkpoint inhibitor is administered in an amount of about 2 mg/kg to 15 mg/kg. A method for treating and/or preventing cancer according to any one of claims 1 to 17, wherein the subject has a cancer selected from hepatocellular carcinoma, colorectal cancer, renal cell carcinoma, bladder cancer, lung cancer (including non-small cell lung cancer), gastric cancer, esophageal cancer, sarcoma, mesothelioma, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, uterine cervix, and liver cancer. A method for treating and/or preventing cancer, wherein the subject has renal cell carcinoma in claim 18. A method for treating and/or preventing cancer according to any one of claims 1 to 19, wherein the subject has failed at least one previous chemotherapy or immunotherapy treatment. A method for treating and/or preventing cancer, wherein the subject has a cancer that is refractory to immune checkpoint inhibitor therapy, and preferably, the cancer is resistant to treatment with an anti-PD-1 antibody and/or an anti-CTLA-4 antibody. A method for treating and/or preventing cancer, wherein the subject is identified as a candidate for immune checkpoint inhibitor therapy in claim 21. A method for treating and/or preventing cancer, comprising administering to the subject an additional therapy selected from chemotherapy (alkylating agents, nucleoside analogues, cytoskeletal modifiers, cytostatic agents) and radiotherapy, according to any one of claims 1 to 22. A method for treating and/or preventing cancer, comprising administering to the subject an additional anticancer virus therapy (e.g., rabies virus, Semliki forest fever virus), according to any one of claims 1 to 23. A method for treating and/or preventing cancer according to any one of claims 1 to 24, wherein the subject is a human. A method for treating and/or preventing cancer according to any one of claims 1 to 25, wherein the administration of a first dose of said replicating oncolytic vaccinia virus and a first dose of said immune checkpoint inhibitor to said subject is followed by at least one subsequent sequential simultaneous administration of said virus and said checkpoint inhibitor to said subject. A method for treating and/or preventing cancer, comprising administering to the subject at least first, second and third sequential simultaneous administrations of the replicative anti-cancer vaccinia virus and the checkpoint inhibitor in accordance with claim 26. A method for treating and/or preventing cancer, comprising administering to the subject at least first, second, third and fourth sequential simultaneous administrations of the replicative anti-cancer vaccinia virus and the checkpoint inhibitor in accordance with claim 27. A method for treating and/or preventing cancer according to any one of claims 26 to 28, wherein the subject is administered simultaneously a first dose of the replicative oncolytic vaccinia virus and a first dose of the immune checkpoint inhibitor and at least one subsequent sequential simultaneous administration of the virus and the checkpoint inhibitor, followed by administration to the subject of at least one dose of the checkpoint inhibitor alone. A method for treating and/or preventing cancer according to any one of claims 26 to 29, wherein the sequential simultaneous administration of the preparations comprises an interval of 1 to 3 weeks, preferably an interval of about 1 week, about 2 weeks or about 3 weeks. A method for treating a tumor in a human, comprising the step of co-administering to the human a combination comprising (a) a replicative oncolytic vaccinia virus and (b) an inhibitor of an immune checkpoint protein. A method for treating a tumor, wherein the replicative anti-cancer vaccinia virus in claim 31 is administered intratumorally, intravenously, intraarterially, or intraperitoneally. A method for treating a tumor, wherein the replicative anti-cancer vaccinia virus in claim 31 is administered intratumorally. A method for treating a tumor, wherein the replicative anti-cancer vaccinia virus in claim 31 is administered in an amount effective to induce expression of an immune checkpoint protein in the tumor. A method for treating a tumor in claim 31, wherein the tumor does not express the immune checkpoint protein or expresses the immune checkpoint protein at a relatively low level prior to administering the replicative anti-cancer vaccinia virus. A method for treating a tumor, comprising a step of measuring the expression level of the immune checkpoint protein in the tumor before administering the combination in claim 34 or 35. A method for treating a tumor according to any one of claims 31 to 36, wherein the immune checkpoint protein is selected from PD-1, PD-L1, CTLA-4, LAG3, TIM3, and TTGTT. A method for treating a tumor, wherein the immune checkpoint protein in claim 31 is CTLA-4. A method for treating a tumor, wherein the immune checkpoint protein in claim 31 is PD-L1. A method for treating a tumor, wherein the immune checkpoint protein in claim 31 is LAG3. A method for treating a tumor, wherein the immune checkpoint protein in claim 31 is TIGIT. A method for treating a tumor, wherein the immune checkpoint protein in claim 31 is PD-1. A method for treating a tumor, wherein the immune checkpoint protein in claim 31 is TIM3. A method for treating a tumor according to any one of claims 31 to 43, wherein the tumor is a solid tumor. A method for treating a tumor according to any one of claims 31 to 43, wherein the tumor is colorectal cancer. A method for treating a tumor according to any one of claims 31 to 43, wherein the tumor is renal cell carcinoma. A method for treating a tumor in claim 39 or 42, wherein the inhibitor of the immune checkpoint protein is a monoclonal antibody that selectively binds to PD-1 or PD-L1, preferably selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab and lambrolizumab. A method for treating a tumor in claim 38, wherein the inhibitor of the immune checkpoint protein is a monoclonal antibody that selectively binds to CTLA-4, preferably selected from the group consisting of ipilimumab and tremelimumab. A method for treating a tumor in a human, comprising the step of co-administering to the human a combination comprising (a) a replicative oncolytic vaccinia virus, (b) an inhibitor of PD-1 and/or PD-L1, and (c) an inhibitor of an immune checkpoint protein. A method for treating a tumor in claim 49, wherein the tumor does not express the immune checkpoint protein or expresses the immune checkpoint protein at a relatively low level prior to administering the replicative anti-cancer vaccinia virus. A method for treating a tumor, wherein the replicative anti-cancer vaccinia virus in claim 49 is administered intratumorally, intravenously, intraarterially, or intraperitoneally. A method for treating a tumor, wherein the replicative anti-cancer vaccinia virus in claim 49 is administered intratumorally. A method for treating a tumor, wherein the replicative anti-cancer vaccinia virus in claim 49 is administered in an amount effective to induce expression of an immune checkpoint protein in the tumor. A method for treating a tumor, wherein the immune checkpoint protein is not expressed or is expressed at a relatively low level prior to administering the replicative anti-cancer vaccinia virus in claim 49. A method for treating a tumor, comprising the step of measuring the expression level of the gateway protein in the tumor, in claim 53 or 54. A method for treating a tumor according to any one of claims 49 to 55, wherein the immune checkpoint protein is selected from CTLA-4, LAG3, TIM3 and TIGIT. A method for treating a tumor, wherein the immune checkpoint protein in claim 56 is CTLA-4. A method for treating a tumor, wherein the immune checkpoint protein in claim 56 is LAG3. A method for treating a tumor, wherein the immune checkpoint protein in claim 56 is TIGIT. A method for treating a tumor, wherein the immune checkpoint protein in claim 56 is TIM3. A method for treating a tumor according to any one of claims 49 to 60, wherein the tumor is a solid tumor. A method for treating a tumor according to any one of claims 49 to 61, wherein the tumor is colorectal cancer. A method for treating a tumor according to any one of claims 49 to 62, wherein the tumor is renal cell carcinoma. A method for treating a tumor, wherein in claim 49, the inhibitor of the immune checkpoint protein is a monoclonal antibody that selectively binds to PD-1 or PD-L1, and is preferably selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, and lambrolizumab. A method for treating a tumor, wherein in claim 57, the inhibitor of the immune checkpoint protein is a monoclonal antibody that selectively binds to CTLA-4, preferably selected from the group consisting of ipilimumab and tremelimumab.